Methods and compositions for identifying receptor effectors

ABSTRACT

The present invention makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which induce or antagonize receptor bioactivity. The subject assay is particularly amenable for identifying surrogate ligands for orphan receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/582,333, filed Jan. 17, 1996, which is a continuation-in-part of U.S.Ser. No. 08/322,137, filed Oct. 13, 1994, now pending, which is acontinuation-in-part of U.S. Ser. No. 08/309,313, filed Sep. 20, 1994,now abandoned, which is a continuation-in-part of U.S. Ser. No.08/190,328, filed Jan. 31, 1994, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/041,431, filed Mar. 31, 1993,now abandoned, the specifications of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] A common technique for cloning receptors is to use nucleic acidhybridization technology to identify receptors which are homologous toother, known receptors. For instance, originally the cloning of seventransmembrane domain G protein-coupled receptors (GCR) depended on theisolation and sequencing of the corresponding protein or the use ofexpression cloning techniques. However, when sequences for thesereceptors became available, it was apparent that there were significantsequence homologies between these receptors. This technology, since itdoes not require that the ligand of the receptor have been identified,has resulted in the cloning of a large number of “orphan receptors”,which have no known ligand and often whose biological function isobscure. Receptors of all types comprise this large family. Known orphanreceptors include the nuclear receptors COUP-TF1/EAR3, COUP-TF2/ARP1,EAR-1, EAR-2, TR-2, PPAR1, HNF-4, ERR-1, ERR-2, NGFIB/Nur77, ELP/SF-1and MPL (Parker et al, supra, and Power et al. (1992) TIBS 13:318-323).A large number of orphan receptors have been identified in the EPHfamily (Hirai et al (1987) Science 238:1717-1720). HER3 and HER4 areorphan receptors in the epidermal growth factor receptor family (Plowmanet al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750). ILA is a newlyidentified member of the human nerve growth factor/tumor necrosis factorreceptor family (Schwarz et al. (1993) Gene 134:295-298). IRRR is anorphan insulin receptor-related receptor which is a transmembranetyrosine kinase (Shier et al. (1989) J. Biol Chem 264:14606-14608).Several orphan tyrosine kinase receptors have been found in Drosophila(Perrimon (1994) Curr. Opin. Cell Biol. 6:260-266). The importance ofidentifying ligands for orphan receptors is clear; it opens up a widearea for research in the area of drug discovery.

[0003] One large subgroup of orphan receptors, as alluded to above, arefound in the G protein coupled receptor family. Approximately 100 suchreceptors have been identified by function and these mediatetransmembrane signaling from external stimuli (vision, taste and smell),endocrine function (pituitary and adrenal), exocrine function(pancreas), heart rate, lipolysis, and carbohydrate metabolism.Structural and genetic similarities suggest that G protein-coupledreceptor superfamily can be subclassified into five distinct groups: (i)amine receptors (serotonin, adrenergic, etc.); (ii) small peptidehormone (somatostatin, TRH, etc.); (iii) large peptide hormone (LH-CG,FSH, etc.); (iv) secretin family; and (v) odorant receptors (Buck L. andAxel, R. (1991) Cell 65:175-187), with orphan receptors apparentlyoccurring in each of the sub-families.

[0004] Previous work describes the expression of recombinant mammalian Gprotein-coupled receptors as a means of studying receptor function as ameans of identifying agonists and antagonists of those receptors. Forexample, the human muscarinic receptor (HM1) has been functionallyexpressed in mouse cells (Harpold et al. U.S. Pat. No. 5,401,629). Therat V1b vasopressin receptor has been found to stimulatephosphotidy.inositol hydrolysis and intracellular Ca2+ mobilization inChinese hamster ovary cells upon agonist stimulation (Lolait et al.(1995) Proc Natl. Acad Sci. USA 92:6783-6787). These types of ectopicexpression studies have enabled researchers to study receptor signallingmechanisms and to perform mutagenisis studies which have been useful inidentifying portions of receptors that are critical for ligand bindingor signal transduction.

[0005] Experiments have also been undertaken to express functional Gprotein coupled receptors in yeast cells. For example, U.S. Pat. No.5,482,835 to King et al. describes a transformed yeast cell which isincapable of producing a yeast G protein α subunit, but which has beenengineered to produce both a mammalian G protein α-subunit and amammalian receptor which is “coupled to” (i.e., interacts with) theaforementioned mammalian G protein α-subunit. Specifically, U.S. Pat.No. 5,482,835 reports expression of the human beta-2 adrenergic receptor(β2AR), a seven transmembrane receptor (STR), in yeast, under control ofthe GAL1 promoter, with the β2AR gene modified by replacing the first 63base pairs of coding sequence with 11 base pairs of noncoding and 42base pairs of coding sequence from the STE2 gene. (STE2 encodes theyeast a-factor receptor). The Duke researchers found that the modifiedβ2AR was functionally integrated into the membrane, as shown by studiesof the ability of isolated membranes to interact properly with variousknown agonists and antagonists of β2AR. The ligand binding affinity foryeast-expressed β2AR was said to be nearly identical to that observedfor naturally produced β2AR.

[0006] U.S. Pat. No. 5,482,835 describes co-expression of a rat Gprotein α-subunit in the same cells, yeast strain 8C, which lacks thecognate yeast protein. Ligand binding resulted in G protein-mediatedsignal transduction. U.S. Pat. No. 5,482,835 teaches that these cellsmay be used in screening compounds for the ability to affect the rate ofdissociation of Gα from Gβγ in a cell. For this purpose, the cellfurther contains a pheromone-responsive promoter (e.g. BAR1 or FUS1),linked to an indicator gene (e.g. HIS3 or LacZ). The cells are placed inmulti-titer plates, and different compounds are placed in each well. Thecolonies are then scored for expression of the indicator gene.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a rapid, reliable and effectiveassay for screening and identifying pharmaceutically effective compoundsthat specifically interact with and modulate the activity of a cellularreceptor or ion channel. The subject assay enables rapid screening oflarge numbers of polypeptides in a library to identifying thosepolypeptides which agonize or antagonize receptor bioactivity. Ingeneral, the assay is characterized by the use of a library ofrecombinant cells, each cell of which include (i) a target receptorprotein whose signal transduction activity can be modulated byinteraction with an extracellular signal, the transduction activitybeing able to generate a detectable signal, and (ii) an expressiblerecombinant gene encoding an exogenous test polypeptide from apolypeptide library. By the use of a variegated gene library, themixture of cells collectively express a variegated population of testpolypeptides. In preferred embodiments, the polypeptide library includesat least 10³ different polypeptides, though more preferably at least10⁵, 10⁶, or 10⁷ different (variegated) polypeptides. The polypeptidelibrary can be generated as a random peptide library, as a semi-randompeptide library (e.g., based on combinatorial mutagenesis of a knownligand), or as a cDNA library.

[0008] The ability of particular constituents of the peptide library tomodulate the signal transduction activity of the target receptor can bescored for by detecting up or down-regulation of the detection signal.For example, second messenger generation via the receptor can bemeasured directly. Alternatively, the use of a reporter gene can providea convenient readout. In any event, a statistically significant changein the detection signal can be used to facilitate isolation of thosecells from the mixture which contain a nucleic acid encoding a testpolypeptide which is an effector of the target receptor.

[0009] By this method, test polypeptides which induce receptor signalingcan be identified. If the test polypeptide does not appear to directlyinduce the activity of the receptor protein, the assay may be repeatedand modified by the introduction of a step in which the recombinant cellis first contacted with a known activator of the target receptor toinduce the signal transdution pathways from the receptor. In oneembodiment, the test polypeptide is assayed for its ability toantagonize, e.g., inhibit or block the activity of the activator.Alternatively, the assay can score for peptides from the peptide librarywhich potentiate the induction response generated by treatment of thecell with a known activator. As used herein, an “agonist” refers toagents which either induce activation of receptor signalling pathways,e.g., such as by mimicking a ligand for the receptor, as well as agentswhich potentiate the sensitivity of the receptor to a ligand, e.g.,lower the concentrations of ligand required to induce a particular levelof receptor-dependent signalling.

[0010] In one embodiment of the present invention the reagent cellsexpress the receptor of interest endogenously. In yet other embodiments,the cells are engineered to express a heterlogous target receptorprotein. In either of these embodiments, it may be desirable toinactivate one or more endogenous genes of the host cells. For example,certain preferred embodiments in which a heterlogous receptor isprovided utilize host cells in which the gene for the homologousreceptor has been inactivated. Likewise, other proteins involved intransducing signals from the target receptor can be inactivated, orcomplemented with an ortholog or paralog from another organism, e.g.,yeast G protein subunits can be complemented by mammalian G proteinsubunits in yeast cells also engineered to express a mammalian G proteincoupled receptor. Other complementations include, for example,expression -of heterologous MAP kinases or erk kinases, MEKs or MKKs(MAP kinase kinases), MEKKs (MEK kinases), ras, raf, STATs, JAKs and thelike.

[0011] The receptor protein can be any receptor which interacts with anextracellular molecule (i.e. hormone, growth factor, peptide) tomodulate a signal in the cell. To illustrate the receptor can be a cellsurface receptor, or in other embodiments can be an intracellularreceptor. In preferred embodiments, the receptor is a cell surfacereceptor, such as: a receptor tyrosine kinase, e.g., an EPH receptor; anion channel; a cytokine receptor; an multisubunit immune recognitionreceptor, a chemokine receptor; a growth factor receptor, or a G-proteincoupled receptor, such as a chemoattracttractant peptide receptor, aneuropeptide receptor, a light receptor, a neurotransmitter receptor, ora polypeptide hormone receptor.

[0012] Preferred G protein coupled receptors include α1A-adrenergicreceptor, α1B-adrenergic receptor, α2-adrenergic receptor,α2B-adrenergic receptor, β1-adrenergic receptor, β2-adrenergic receptor,β3-adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR, m3AChR, m4 AChR, m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, Aladenosine receptor, A2b adenosine receptor, 5-HT1a receptor, 5-HT1breceptor, 5HT1-like receptor, 5-HT1d receptor, 5HT1d-like receptor,5HT1d beta receptor, substance K (neurokinin A) receptor, fMLP receptor,fMLP-like receptor, angiotensin II type 1 receptor, endothelin ETAreceptor, endothelin ETB receptor, thrombin receptor, growthhormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptidereceptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3,cannabinoid receptor, follicle stimulating hormone (FSH) receptor,leutropin (LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5aanaphylatoxin receptor, Interleukin 8 (IL-8) IL-8RA, IL-8RB, DeltaOpioid receptor, Kappa Opioid receptor, mip-1/RANTES receptor,Rhodopsin, Red opsin, Green opsin, Blue opsin, metabotropic glutamatemGluR1-6, histamine H2 receptor, ATP receptor, neuropeptide Y receptor,amyloid protein precursor receptor, insulin-like growth factor IIreceptor, bradykinin receptor, gonadotropin-releasing hormone receptor,cholecystokinin receptor, melanocyte stimulating hormone receptorreceptor, antidiuretic hormone receptor, glucagon receptor, andadrenocorticotropic hormone II receptor.

[0013] Preferred EPH receptors inlcude eph, elk, eck, sek, mek4, hek,hek2, eek, erk, tyro1, tyro4, tyro5, tyro6, tyro11, cek4, cek5, cek6,cek7, cek8, cek9, cek10, bsk, rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2,pagliaccio, htk, erk and nuk receptors.

[0014] As set forth below, no matter which structural/function class towhich the target receptor may belong, the subject assay is amenable toidentifying ligands for an otherwise orphan receptor.

[0015] In those embodiments wherein the target receptor is a cellsurface receptor, it will be desirable for the peptides in the libraryto express a signal sequence to ensure that they are processed in theappropriate secretory pathway and thus are available to interact withreceptors on the cell surface.

[0016] With respect to a detection signal generated by signaltransduction, certain of the preferred embodiments measure theproduction of second messengers to determine changes in ligandengagement by the receptor. In preferred embodiments, changes in GTPhydrolysis, calcium mobilization, or phospholipid hydrolysis can bemeasured.

[0017] In other preferred embodiment, the host cells harbors a reporterconstruct containing a reporter gene in operative linkage with one ormore transcriptional regulatory elements responsive to the signaltransductin activity of the receptor protein. Exemplary reporter genesinclude enzymes, such as luciferase, phosphatase, or β-galactosidasewhich can produce a spectrometrically active label, e.g., changes incolor, fluorescence or luminescence, or a gene product which alters acellular phenotype, e.g., cell growth, drug resistance or auxotrophy. Inpreferred embodiments: the reporter gene encodes a gene product selectedfrom the group consisting of chloramphenicol acetyl transferase,beta-galactosidase and secreted alkaline phosphatase; the reporter geneencodes a gene product which confers a growth signal; the reporter geneencodes a gene product for growth in media containing aminotriazole orcanavanine.

[0018] The reagent cells of the present invention can be derived fromany eukaryotic organism. In preferred embodiments the cells aremammalian cells. In more preferred embodiments the cells are yeastcells, with cells from the genera Saccharomyces or Schizosaccharomycesbeing more preferred. However, cells from amphibia (such as xenopus),avian or insect sources are also contemplated. The host cells canderived from primary cells, or transformed and/or immortalized celllines.

[0019] In another aspect, the present invention provides

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Structures of pAAH5 andpRS-ADC.

[0020]FIG. 2. Schematic diagram of the structure of the plasmidresulting from insertion of random oligonucleotides into pADC-MF alpha.This plasmid expresses random peptides in the context of the MF alpha 1signal and leader peptide.

[0021]FIG. 3. Schematic diagram of the structure of the plasmidresulting from insertion of random oligonucleotides into pADC-MFa. Thisplasmid expresses random peptides in the context of the MFa1 leader andC-terminal CVIA tetrapeptide.

[0022]FIG. 4. Activity of a fus1 promoter in response to signaling byhuman C5a expressed in autocrine strains of yeast.

[0023]FIG. 5. Exemplary set of steps for isolating surrogate ligands forthe C5a receptor.

[0024]FIG. 6. Spotting a lawn of recombinant yeast cells with variousC5a receptor agonists or DMF solvent control.

[0025]FIG. 7. The amino acid sequence for C5a surrogate agonistpeptides.

[0026]FIG. 8. Dose response curve for various C5a receptor surrogatepeptide ligands based on a colorimetric lacZ readout.

[0027]FIG. 9. Expression of a lacZ reporter gene construct, engineeredinto the mammalian HEK293 cell-line, in response to stimulation of a C5areceptor by a C5a receptor agonist.

[0028]FIG. 10. Dose-response curves comparing a second generation C5areceptor agonist (122mod1-5) with other known C5a receptor agonsts.

[0029]FIG. 11. Autocrine activation of the pheromone response pathway inyeast expressing FPRL-1 agonists or C5a receptor agonists.

[0030]FIG. 12. Intracellular Ca⁺⁺ mobilization in neutrophils asdetected by fluorescence activated Cell Sorter analysis using FURA2 dyeabsorbance ratio. The measurements were performed for the C5a peptide,or no peptide (control), or varying concentrations of the A5 peptide.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Proliferation, differentiation and death of eukaryotic cells arecontrolled by hormones, neurotransmitters, and polypeptide factors.These diffusible ligands allow cells to influence and be influenced byenvironmental cues. The study of receptor-ligand interaction hasrevealed a great deal of information about how cells respond to externalstimuli, and this knowledge has led to the development oftherapeutically important compounds. However, the rate at whichreceptors have been cloned has recently increased dramatically—existingfamilies have been extended and new families recognized. In particular,the application of advanced cloning approaches has allowed the isolationof many receptors for which ligands are initially unknown. These arecommonly referred to in the art as “orphan” receptors, and several havesubsequently proved to be important pharmacological targets.

[0032] The present invention makes available a rapid, effective assayfor screening and identifying pharmaceutically effective compounds thatspecifically interact with and modulate the activity of a cellularreceptor or ion channel. The subject assay enables rapid screening oflarge numbers of polypeptides in a library to identifying thosepolypeptides which induce or antagonize receptor bioactivity.

[0033] In general, the assay is characterized by the use of a mixture ofrecombinant cells to sample a variegated polypeptide library forreceptor agonists or antagonists. As described with greater detailbelow, the reagent cells express both a target receptor protein capableof transducing a detectable signal in the reagent cell, and a testpolypeptide for which interaction with the receptor is to beascertained. Collectively, a culture of such reagent cells will providea variegated library of potential receptor effectors and those membersof the library which either agonize or antagonize the receptor functioncan be selected and identified by sequence.

[0034] One salient feature of the subject assay is the enhancedsensitivity resulting from expression of the test polypeptide in a cellwhich also serves as a reporter for the desired receptor-ligandinteraction. To illustrate, where the detectable signal resulting fromreceptor engagement by an agonist provides a growth signal or drugresistance, individual cells expressing polypeptides which agonizereceptor function can be amplified and isolated from a library culture.

[0035] Accordingly, the present invention provides a convenient formatfor discovering drugs which can be useful to modulate cellular function,as well as to understand the pharmacology of compounds that specificallyinteract with cellular receptors or ion channels. Moreover, the subjectassay is particularly amenable to identifying ligands, natural orartifical, for orphan receptors.

[0036] Before further description of the invention, certain termsemployed in the specification, examples and appended claims are, forconvenience, collected here.

[0037] As used herein, “recombinant cells” include any cells that havebeen modified by the introduction of heterologous DNA. Control cellsinclude cells that are substantially identical to the recombinant cells,but do not express one or more of the proteins encoded by theheterologous DNA, e.g., do not include or express the reporter geneconstruct, receptor or test polypeptide.

[0038] The terms “recombinant protein”, “heterologous protein” and“exogenous protein” are used interchangeably throughout thespecification and refer to a polypeptide which is produced byrecombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.That is, the polypeptide is expressed from a heterologous nucleic acid.

[0039] As used herein, “heterologous DNA” or “heterologous nucleic acid”include DNA that does not occur naturally as part of the genome in whichit is present or which is found in a location or locations in the genomethat differs from that in which it occurs in nature. Heterologous DNA isnot endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchDNA encodes RNA and proteins that are not normally produced by the cellin which it is expressed. Heterologous DNA may also be referred to asforeign DNA. Any DNA that one of skill in the art would recognize orconsider as heterologous or foreign to the cell in which is expressed isherein encompassed by heterologous DNA. Examples of heterologous DNAinclude, but are not limited to, DNA that encodes test polypeptides,receptors, reporter genes, transcriptional and translational regulatorysequences, selectable or traceable marker proteins, such as a proteinthat confers drug resistance.

[0040] As used herein, “cell surface receptor” refers to molecules thatoccur on the surface of cells, interact with the extracellularenvironment, and transmit or transduce the information regarding theenvironment intracellularly in a manner that ultimately modulatestranscription of specific promoters, resulting in transcription ofspecific genes.

[0041] As used herein, “extracellular signals” include a molecule or achange in the environment that is transduced intracellularly via cellsurface proteins that interact, directly or indirectly, with the signal.An extracellular signal or effector molecule includes any compound orsubstance that in some manner specifically alters the activity of a cellsurface protein. Examples of such signals include, but are not limitedto, molecules such as acetylcholine, growth factors and hormones, thatbind to cell surface and/or intracellular receptors and ion channels andmodulate the activity of such receptors and channels.

[0042] As used herein, “extracellular signals” also include as yetunidentified substances that modulate the activity of a cellularreceptor, and thereby influence intracellular functions. Suchextracellular signals are potential pharmacological agents that may beused to treat specific diseases by modulating the activity of specificcell surface receptors.

[0043] “Orphan receptors” is a designation given to a receptors forwhich no specific natural ligand has been described.

[0044] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to a transcriptionalregulatory sequences. Transcription of the reporter gene is controlledby these sequences. The activity of at least one or more of thesecontrol sequences is directly or indirectly regulated by the targetreceptor protein. The transcriptional regulatory sequences include thepromoter and other regulatory regions, such as enhancer sequences, thatmodulate the activity of the promoter, or regulatory sequences thatmodulate the activity or efficiency of the RNA polymerase thatrecognizes the promoter, or regulatory sequences are recognized byeffector molecules, including those that are specifically induced byinteraction of an extracellular signal with the target receptor. Forexample, modulation of the activity of the promoter may be effected byaltering the RNA polymerase binding to the promoter region, or,alternatively, by interfering with initiation of transcription orelongation of the mRNA. Such sequences are herein collectively referredto as transcriptional regulatory elements or sequences. In addition, theconstruct may include sequences of nucleotides that alter translation ofthe resulting mRNA, thereby altering the amount of reporter geneproduct.

[0045] “Signal transduction” is the processing of chemical signals fromthe cellular environment through the cell membrane, and may occurthrough one or more of several mechanisms, such as phosphorylation,activation of ion channels, effector enzyme activation via guaninenucleotide binding protein intermediates, formation of inositolphosphate, activation of adenylyl cyclase, and/or direct activation (orinhibition) of a transcriptional factor.

[0046] The term “modulation of a signal transduction activity of areceptor protein” in its various grammatical forms, as used herein,designates induction and/or potentiation, as well as inhibition of oneor more signal transduction pathways downstream of a receptor.

[0047] Agonists and antagonists are “receptor effector” molecules thatmodulate signal transduction via a receptor. Receptor effector moleculesare capable of binding to the receptor, though not necessarily at thebinding site of the natural ligand. Receptor effectors can modulatesignal transduction when used alone, i.e. can be surrogate ligands, orcan alter signal transduction in the presence of the natural ligand,either to enhance or inhibit signaling by the natural ligand. Forexample, “antagonists” are molecules that block or decrease the signaltransduction activity of receptor, e.g., they can competitively,noncompetitively, and/or allosterically inhibit signal transduction fromthe receptor, whereas “agonists” potentiate, induce or otherwise enhancethe signal transduction activity of a receptor. The terms “receptoractivator” and “surrogate ligand” refer to an agonist which inducessignal transduction from a receptor.

[0048] The term “substantially homologous”, when used in connection withamino acid sequences, refers to sequences which are substantiallyidentical to or similar in sequence, giving rise to a homology inconformation and thus to similar biological activity. The term is notintended to imply a common evolution of the sequences.

[0049] Typically, “substantially homologous” sequences are at least 50%,more preferably at least 80%, identical in sequence, at least over anyregions known to be involved in the desired activity. Most preferably,no more than five residues, other than at the termini, are different.Preferably, the divergence in sequence, at least in the aforementionedregions, is in the form of “conservative modifications”.

[0050] The term “autocrine cell”, as used herein, refers to a cell whichproduces a substance which can stimulate a receptor located on or withinthe same cell as produces the substance. For example, wild-type yeast αand a cells are not autocrine. However, a yeast cell which produces bothα-factor and α-factor receptor, or both α-factor and a-factor receptor,in functional form, is autocrine. By extension, cells which produce apeptide which is being screened for the ability to activate a receptor(e.g., by activating a G protein-coupled receptor) express the receptorare called “autocrine cells”, though it might be more precise to callthem “putative autocrine cells”. Of course, in a library of such cells,in which a multitude of different peptides are produced, it is likelythat one or more of the cells will be “autocrine” in the stricter senseof the term.

[0051] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein.

[0052] I. Overview of Assay

[0053] As set out above, the present invention relates to methods foridentifying effectors of a receptor protein or complex thereof. Ingeneral, the assay is characterized by the use of a library ofrecombinant cells, each cell of which include (i) a target receptorprotein whose signal transduction activity can be modulated byinteraction with an extracellular signal, the transduction activitybeing able to generate a detectable signal, and (ii) an expressiblerecombinant gene encoding an exogenous test polypeptide from apolypeptide library. By the use of a variegated gene library, themixture of cells collectively express a variegated population of testpolypeptides.

[0054] The ability of particular constituents of the peptide library tomodulate the signal transduction activity of the target receptor can bescored for by detecting up or down-regulation of the detection signal.For example, second messenger generation (e.g. GTPase activity,phospholipid hydrolysis, or protein phosphorylation) via the receptorcan be measured directly. Alternatively, the use of a reporter gene canprovide a convenient readout. In any event, a statistically significantchange in the detection signal can be used to facilitate isolation ofthose cells from the mixture which contain a nucleic acid encoding atest polypeptide which is an effector of the target receptor.

[0055] By this method, test polypeptides which induce the receptor'ssignaling can be screened. If the test polypeptide does not appear toinduce the activity of the receptor protein, the assay may be repeatedand modified by the introduction of a step in which the recombinant cellis first contacted with a known activator of the target receptor toinduce signal transduction from the receptor, and the test polypeptideis assayed for its ability to inhibit the activity of the receptor,e.g., to identify receptor antagonists. In yet other embodiments, thepeptide library can be screened for members which potentiate theresponse to a known activator of the receptor. In this respect,surrogate ligands identified by the present assay for orphan receptorscan be used as the exogenous activator, and further peptide librariesscreened for members which potentiate or inhibit the activating peptide.Alternatively, the surrogate ligand can be used to screen exogenouscompound libraries (peptide and non-peptide) which, by modulating theactivity of the identified surrogate, will presumably also similarlyeffect the native ligand's effect on the target receptor. In suchembodiments, the surrogate ligand can be applied to the cells, though ispreferably produced by the reagent cell, thereby providing an autocrinecell.

[0056] In developing the recombinant cells assays, it was recognizedthat a frequent result of receptor-mediated responses to extracellularsignals was the transcriptional acitivation or inactivation of specificgenes after exposure of the cognate receptor to an extracellular signalthat induces such activity. Thus, transcription of genes controlled byreceptor-responsive transcriptional elements often reflects the activityof the surface protein by virtue of transduction of an intracellularsignal.

[0057] To illustrate, the intracellular signal that is transduced can beinitiated by the specific interaction of an extracellular signal,particularly a ligand, with a cell surface receptor on the cell. Thisinteraction sets in motion a cascade of intracellular events, theultimate consequence of which is a rapid and detectable change in thetranscription or translation of a gene. By selecting transcriptionalregulatory sequences that are responsive to the transduced intracellularsignals and operatively linking the selected promoters to reportergenes, whose transcription, translation or ultimate activity is readilydetectable and measurable, the transcription based assay provides arapid indication of whether a specific receptor or ion channel interactswith a test peptide in any way that influences intracellulartransduction. Expression of the reporter gene, thus, provides a valuablescreening tool for the development of compounds that act as agonists orantagonists of a cell receptor or ion channel.

[0058] Reporter gene based assays of this invention measure the endstage of the above described cascade of events, e.g., transcriptionalmodulation. Accordingly, in practicing one embodiment of the assay, areporter gene construct is inserted into the reagent cell in order togenerate a detection signal dependent on receptor signaling. Typically,the reporter gene construct will include a reporter gene in operativelinkage with one or more transcriptional regulatory elements responsiveto the signal transduction activity of the target receptor, with thelevel of expression of the reporter gene providing thereceptor-dependent detection signal. The amount of transcription fromthe reporter gene may be measured using any method known to those ofskill in the art to be suitable. For example, specific mRNA expressionmay be detected using Northern blots or specific protein product may beidentified by a characteristic stain or an intrinsic activity.

[0059] In preferred embodiments, the gene product of the reporter isdetected by an intrinsic activity associated with that product. Forinstance, the reporter gene may encode a gene product that, by enzymaticactivity, gives rise to a detection signal based on color, fluorescence,or luminescence.

[0060] The amount of expression from the reporter gene is then comparedto the amount of expression in either the same cell in the absence ofthe test compound or it may be compared with the amount of transcriptionin a substantially identical cell that lacks the specific receptors. Acontrol cell may be derived from the same cells from which therecombinant cell was prepared but which had not been modified byintroduction of heterologous DNA, e.g., the encoding the testpolypeptide. Alternatively, it may be a cell in which the specificreceptors are removed. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testpolypeptide has in some manner altered the activity of the specificreceptor.

[0061] In other preferred embodiments, the reporter or marker geneprovides a selection method such that cells in which the peptide is aligand for the receptor have a growth advantage. For example thereporter could enhance cell viability, relieve a cell nutritionalrequirement, and/or provide resistance to a drug.

[0062] With respect to the target receptor, it may be endogenouslyexpressed by the host cell, or it may be expressed from a heterologousgene that has been introduced into the cell. Methods for introducingheterologous DNA into eukaryotic cells are of course well known in theart and any such method may be used. In addition, DNA encoding variousreceptor proteins is known to those of skill in the art or it may becloned by any method known to those of skill in the art. In certainembodiments, such as when an exogenous receptor is expressed, it may bedesirable to inactivate, such as by deletion, a homologous receptorpresent in the cell.

[0063] The subject assay is useful for identifying polypeptides thatinteract with any receptor protein whose activity ultimately induces asignal transduction cascade in the host cell which can be exploited toproduce a detectable signal. In particular, the assays can be used totest functional ligand-receptor or ligand-ion channel interactions forcell surface-localized receptors and channels, and also for cytoplasmicand nuclear receptors. As described in more detail below, the subjectassay can be used to identify effectors of, for example, Gprotein-coupled receptors, receptor tyrosine kinases, cytokinereceptors, and ion channels, as well as steroid hormone receptors. Inpreferred embodiments the method described herein is used foridentifying ligands for “orphan receptors” for which no ligand is known.

[0064] In embodiments in which cell surface receptors are the assaytargets, it will be desirable for each of the peptides of the peptidelibrary to include a signal sequence for secretion, e.g., which willensure appropriate transport of the peptide to the endoplasmicreticulum, the golgi, and ultimately to the cell surface so that it isable to interact with cell surface receptors. In the case of yeastcells, the signal sequence will transport peptides to the periplasmicspace.

[0065] Any transfectable cell that can express the desired cell surfaceprotein in a manner such the protein functions to intracellularlytransduce an extracellular signal may be used. The cells may be selectedsuch that they endogenously express the target receptor protein or maybe genetically engineered to do so.

[0066] The preparation of cells which express the orphan FPRL1 receptor,a peptide library, and a reporter gene expression construct, aredescribed. These cells have been used to identify a novel ligand forthis receptor. The cells for the identification of receptor ligands andin drug screening assays to discover agents capable of modulatingreceptor activity.

[0067] Any cell surface protein that is known to those of skill in theart or that may be identified by those of skill in the art may used inthe assay. The cell surface protein may endogenously expressed on theselected cell or it may be expressed from cloned DNA.

[0068] II. Host Cells

[0069] Suitable host cells for generating the subject assay includeprokaryotes, yeast, or higher eukaryotic cells, especially mammaliancells. Prokaryotes include gram negative or gram positive organisms.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman (1981) Cell 23:175) CV-1cells (ATCC CCL 70), L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines.

[0070] If yeast cells are used, the yeast may be of any species whichare cultivable and in which an exogenous receptor can be made to engagethe appropriate signal transduction machinery of the host cell. Suitablespecies include Kluyverei lactis, Schizosaccharomyces pombe, andUstilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeastwhich can be used in practicing the present invention are Neurosporacrassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris,Candida tropicalis, and Hansenula polymorpha. The term “yeast”, as usedherein, includes not only yeast in a strictly taxonomic sense, i.e.,unicellular organisms, but also yeast-like multicellular fungi orfilamentous fungi.

[0071] The choice of appropriate host cell will also be influenced bythe choice of detection signal. For instance, reporter constructs, asdescribed below, can provide a selectable or screenable trait upontranscriptional activation (or inactivation) in response to a signaltransduction pathway coupled to the target receptor. The reporter genemay be an unmodified gene already in the host cell pathway, such as thegenes responsible for growth arrest in yeast. It may be a host cell genethat has been operably linked to a “receptor-responsive” promoter.Alternatively, it may be a heterologous gene that has been so linked.Suitable genes and promoters are discussed below. In other embodiments,second messenger generation can be measured directly in the detectionstep, such as mobilization of intracellular calcium or phospholipidmetabolism are quantitated. Accordingly, it will be understood that toachieve selection or screening, the host cell must have an appropriatephenotype. For example, introducing a pheromone-responsive chimeric HIS3gene into a yeast that has a wild-type HIS3 gene would frustrate geneticselection. Thus, to achieve nutritional selection, an auxotrophic strainis wanted.

[0072] To further illustrate, in a preferred embodiment of the subjectassay using a yeast host cell, the yeast cells possess one or more ofthe following characteristics: (a) the endogenous FUS1 gene has beeninactivated; (b) the endogenous SST2 gene, and/or other genes involve indesensitization, has been inactivated; (c) if there is a homologous,endogenous receptor gene it has been inactivated; and (d) if the yeastproduces an endogenous ligand to the exogenous receptor, the genesencoding for the ligand been inactivated.

[0073] Other complementations for use in the subject assay can beconstructed without any undue experimentation. Indeed, many yeastgenetic complementation with mammalian signal transduction proteins havebeen described in the art. For example, Mosteller et al. (1994) Mol CellBiol 14:1104-12 demonstrates that human Ras proteins can complement lossof ras mutations in S. cerevisiae. Moreover, Toda et al. (1986) PrincessTakamatsu Symp 17: 253-60 have shown that human ras proteins cancomplement the loss of RAS1 and RAS2 proteins in yeast, and hence arefunctionally homologous. Both human and yeast RAS proteins can stimulatethe magnesium and guanine nucleotide-dependent adenylate cyclaseactivity present in yeast membranes. Ballester et al. (1989) Cell 59:681-6 describe a vector to express the mammalian GAP protein in theyeast S. cerevisiae. When expressed in yeast, GAP inhibits the functionof the human ras protein, and complements the loss of IRA1. IRA1 is ayeast gene that encodes a protein with homology to GAP and acts upstreamof RAS. Mammalian GAP can therefore function in yeast and interact withyeast RAS. Wei et al. (1994) Gene 151: 279-84 describes that a humanRas-specific guanine nucleotide-exchange factor, Cdc25GEF, cancomplement the loss of CDC25 function in S. cerevisiae. Martegani et al.(1992) EMBO J 11: 2151-7 describe the cloning by functionalcomplementation of a mouse cDNA encoding a homolog of CDC25, aSaccharomyces cerevisiae RAS activator. Vojtek et al. (1993) J Cell Sci105: 777-85 and Matviw et al. (1992) Mol Cell Biol 12: 5033-40 describehow a mouse CAP protein, e.g., an adenylyl cyclase associated proteinassociated with ras-mediated signal transduction, can complementsdefects in S. cerevisiae. Papasavvas et al. (1992) Biochem Biophys ResCommun 184:1378-85 also suggest that inactivated yeast adenyl cyclasecan be complemented by a mammalian adenyl cyclase gene. Hughes et al.(1993) Nature 364: 349-52 describe the complementation of byr1 infission yeast by mammalian MAP kinase kinase (MEK). Parissenti et al.(1993) Mol Cell Endocrinol 98: 9-16 describes the reconstitution ofbovine protein kinase C (PKC) in yeast. The Ca(2+)- andphospholipid-dependent Ser/Thr kinase PKC plays important roles in thetransduction of cellular signals in mammalian cells. Marcus et al.(1995) PNAS 92: 6180-4 suggests the complementation of shk1 nullmutations in S. pombe by the either the structurally related S.cerevisiae Ste20 or mammalian p65PAK protein kinases.

[0074] “Inactivation”, with respect to genes of the host cell, meansthat production of a functional gene product is prevented or inhibited.Inactivation may be achieved by deletion of the gene, mutation of thepromoter so that expression does not occur, or mutation of the codingsequence so that the gene product is inactive. Inactivation may bepartial or total.

[0075] “Complementation”, with respect to genes of the host cell, meansthat at least partial function of inactivated gene of the host cell issupplied by an exogenous nucleic acid. For instance, yeast cells can be“mammalianized”, and even “humanized”, by complementation of receptorand signal transduction proteins with mammalian homologs. To illustrate,inactivation of a yeast Byr2/Ste11 gene can be complemented byexpression of a human MEKK gene.

[0076] III. Expression Systems

[0077] Ligating a polynucleotide coding sequence into a gene construct,such as an expression vector, and transforming or transfecting intohosts, either eukaryotic (yeast, avian, insect or mammalian) orprokaryotic (bacterial cells), are standard procedures used in producingother well-known proteins, including sequences encoding exogenousreceptor and peptide libraries. Similar procedures, or modificationsthereof, can be employed to prepare recombinant reagent cells of thepresent invention by tissue-culture technology in accord with thesubject invention.

[0078] In general, it will be desirable that the vector be capable ofreplication in the host cell. It may be a DNA which is integrated intothe host genome, and thereafter is replicated as a part of thechromosomal DNA, or it may be DNA which replicates autonomously, as inthe case of a plasmid. In the latter case, the vector will include anorigin of replication which is functional in the host. In the case of anintegrating vector, the vector may include sequences which facilitateintegration, e.g., sequences homologous to host sequences, or encodingintegrases.

[0079] Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are known in theart, and are described in, for example, Powels et al. (Cloning Vectors:A Laboratory Manual, Elsevier, New York, 1985). Mammalian expressionvectors may comprise non-transcribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5′ or 3′ flanking nontranscribed sequences, and 5′or 3′ nontranslated sequences, such as necessary ribosome binding sites,a poly-adenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

[0080] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0081] The transcriptional and translational control sequences inexpression vectors to be used in transforming mammalian cells may beprovided by viral sources. For example, commonly used promoters andenhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40(SV40), and human cytomegalovirus. DNA sequences derived from the SV40viral genome, for example, SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites may be used to provide theother genetic elements required for expression of a heterologous DNAsequence. The early and late promoters are particularly useful becauseboth are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication (Fiers et al. (1978)Nature 273:111) Smaller or larger SV40 fragments may also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the viral origin of replication isincluded. Exemplary vectors can be constructed as disclosed by Okayamaand Berg (1983, Mol. Cell Biol. 3:280). A useful system for stable highlevel expression of mammalian receptor cDNAs in C127 murine mammaryepithelial cells can be constructed substantially as described by Cosmanet al (1986, Mol. Immunol. 23:935). Other expression vectors for use inmammalian host cells are derived from retroviruses.

[0082] In other embodiments, the use of viral transfection can providestably integrated copies of the expression construct. In particular, theuse of retroviral, adenoviral or adeno-associated viral vectors iscontemplated as a means for providing a stably transfected cell linewhich expresses an exogenous receptor, and/or a polypeptide library.

[0083] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. Moreover, if yeast are used as a host cell, it will be understoodthat the expression of a gene in a yeast cell requires a promoter whichis functional in yeast. Suitable promoters include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Req. 7, 149 (1968); and Holland et al. Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose and galactose utilization. Finally,promoters that are active in only one of the two haploid mating typesmay be appropriate in certain circumstances. Among thesehaploid-specific promoters, the pheromone promoters MFa1 and MFα1 are ofparticular interest.

[0084] In some instances, it may be desirable to derive the host cellusing insect cells. In such embodiments, recombinant polypeptides can beexpressed by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWI),and pBlueBac-derived vectors (such as the B-gal containing pBlueBacIII).

[0085] Libraries of random peptides or cDNA fragments may be expressedin a multiplicity of ways, including as portions of chimeric proteins.As described below, where secretion of the peptide library is desired,the peptide library can be engineered for secretion or transport to theextracellular space via the yeast pheromone system

[0086] In constructing suitable expression plasmids, the terminationsequences associated with these genes, or with other genes which areefficiently expressed in yeast, may also be ligated into the expressionvector 3′ of the heterologous coding sequences to providepolyadenylation and termination of the mRNA.

[0087] IV. Periplasmic Secretion

[0088] If yeast cells are used as the host cell it will be noted thatthe yeast cell is bounded by a lipid bilayer called the plasma membrane.Between this plasma membrane and the cell wall is the periplasmic space.Peptides secreted by yeast cells cross the plasma membrane through avariety of mechanisms and thereby enter the periplasmic space. Thesecreted peptides are then free to interact with other molecules thatare present in the periplasm or displayed on the outer surface of theplasma membrane. The peptides then either undergo re-uptake into thecell, diffuse through the cell wall into the medium, or become degradedwithin the periplasmic space.

[0089] The test polypeptide library may be secreted into the periplasmby any of a number of exemplary mechanisms, depending on the nature ofthe expression system to which they are linked. In one embodiment, thepeptide may be structurally linked to a yeast signal sequence, such asthat present in the α-factor precursor, which directs secretion throughthe endoplasmic reticulum and Golgi apparatus. Since this is the sameroute that the receptor protein follows in its journey to the plasmamembrane, opportunity exists in cells expressing both the receptor andthe peptide library for a specific peptide to interact with the receptorduring transit through the secretory pathway. This has been postulatedto occur in mammalian cells exhibiting autocrine activation. Suchinteraction could yield activation of the response pathway duringtransit, which would still allow identification of those cellsexpressing a peptide agonist. For situations in which peptideantagonists to externally applied receptor agonist are sought, thissystem would still be effective, since both the peptide antagonist andreceptor would be delivered to the outside of the cell in concert. Thus,those cells producing an antagonist would be selectable, since thepeptide antagonist would be properly and timely situated to prevent thereceptor from being stimulated by the externally applied agonist.

[0090] An alternative mechanism for delivering peptides to theperiplasmic space is to use the ATP-dependent transporters of theSTE6/MDR1 class. This transport pathway and the signals that direct aprotein or peptide to this pathway are not as well characterized as isthe endoplasmic reticulum-based secretory pathway. Nonetheless, thesetransporters apparently can efficiently export certain peptides directlyacross the plasma membrane, without the peptides having to transit theER/Golgi pathway. It is anticipated that at least a subset of peptidescan be secreted through this pathway by expressing the library incontext of the a-factor prosequence and terminal tetrapeptide. Thepossible advantage of this system is that the receptor and peptide donot come into contact until both are delivered to the external surfaceof the cell. Thus, this system strictly mimics the situation of anagonist or antagonist that is normally delivered from outside the cell.Use of either of the described pathways is within the scope of theinvention.

[0091] The present invention does not require periplasmic secretion, or,if such secretion is provided, any particular secretion signal ortransport pathway.

[0092] V. Cytokine Receptors

[0093] In one embodiment the target receptor is a cytokine receptor.Cytokines are a family of soluble mediators of cell-to-cellcommunication that includes interleukins, interferons, andcolony-stimulating factors. The characteristic features of cytokines liein their functional redundancy and pleiotropy. Most of the cytokinereceptors that constitute distinct superfamilies do not possessintrinsic protein tyrosine kinase domains, yet receptor stimulationusually invokes rapid tyrosine phosphorylation of intracellularproteins, including the receptors themselves. Many members of thecytokine receptor superfamily acitvate the Jak protein tyrosine kinasefamily, with resultant phosphorylation of the STAT transcriptionalactivator factors. IL-2, IL-7, IL-2 and Interferon γ have all been shownto activate Jak kinases (Frank et al (1995) Proc Natl Acad Sci USA92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon et al.(1995) Proc Natl Acad Sci USA 92:7307-7311); and (Sakatsume et al (1995)J. Biol Chem 270:17528-17534). Events downstream of Jak phosphorylationhave also been elucidated. For example, exposure of T lymphocytes toIL-2 has been shown to lead to the phosphorylation of signal transducersand activators of transcription (STAT) proteins STAT1α, STAT2β, andSTAT3, as well as of two STAT-related proteins, p94 and p95. The STATproteins were found to translocate to the nucleus and to bind to aspecific DNA sequence, thus suggesting a mechanism by which IL-2 mayactivate speicfic genes involved in immune cell function (Frank et al.supra). Jak3 is associated with the gamma chain of the IL-2, IL-4, andIL-7 cytokine receptors (Fujii et al. (1995) Proc Natl Acad Sci92:5482-5486) and (Musso et al (1995) J Exp Med. 181:1425-1431). The Jakkinases have also been shown to be activated by numerous ligands thatsignal via cytokine receptors such as, growth hormone and erythropoietinand IL-6 (Kishimoto (1994) Stem cells Suppl 12:37-44).

[0094] Detection signals which may be scored for in the present assay,in addition to direct detection of second messangers, such as by changesin phosphorylation, includes reporter constructs which includetranscriptional regulatory elements responsive to the STAT proteins.Described infra.

[0095] VI. Multisubunit Immune Recognition Receptor (MIRR).

[0096] In another embodiment the receptor is a multisubunit receptor.Receptors can be comprised of multiple proteins referred to as subunits,one category of which is referred to as a multisubunit receptor is amultisubunit immune recognition receptor (MIRR). MIRRs include receptorshaving multiple noncovalently associated subunits and are capable ofinteracting with src-family tyrosine kinases. MIRRs can include, but arenot limited to, B cell antigen receptors, T cell antigen receptors, Fcreceptors and CD22. One example of an MIRR is an antigen receptor on thesurface of a B cell. To further illustrate, the MIRR on the surface of aB cell comprises membrane-bound immunoglobulin (mIg) associated with thesubunits Ig-α and Ig-β or Ig-γ, which forms a complex capable ofregulating B cell function when bound by antigen. An antigen receptorcan be functionally linked to an amplifier molecule in a manner suchthat the amplifier molecule is capable of regulating gene transcription.

[0097] Src-family tyrosine kinases are enzymes capable ofphosphorylating tyrosine residues of a target molecule. Typically, asrc-family tyrosine kinase contains one or more binding domains and akinase domain. A binding domain of a src-family tyrosine kinase iscapable of binding to a target molecule and a kinase domain is capableof phosphorylating a target molecule bound to the kinase. Members of thesrc family of tyrosine kinases are characterized by an N-terminal uniqueregion followed by three regions that contain different degrees ofhomology among all the members of the family. These three regions arereferred to as src homology region 1 (SH1), src homology region 2 (SH2)and src homology region 3 (SH3). Both the SH2 and SH3 domains arebelieved to have protein association functions important for theformation of signal transduction complexes. The amino acid sequence ofan N-terminal unique region, varies between each src-family tyrosinekinase. An N-terminal unique region can be at least about the first 40amino acid residues of the N-terminal of a src-family tyrosine kinase.

[0098] Syk-family kinases are enzymes capable of phosphorylatingtyrosine residues of a target molecule. Typically, a syk-family kinasecontains one or more binding domains and a kinase domain. A bindingdomain of a syk-family tyrosine kinase is capable of binding to a targetmolecule and a kinase domain is capable of phosphorylating a targetmolecule bound to the kinase. Members of the syk-family of tyrosinekinases are characterized by two SH2 domains for protein associationfunction and a tyrosine kinase domain.

[0099] A primary target molecule is capable of further extending asignal transduction pathway by modifying a second messenger molecule.Primary target molecules can include, but are not limited to,phosphatidylinositol 3-kinase (PI-3K), P21^(ras)GAPase-activatingprotein and associated P190 and P62 protein, phospholipases such asPLCγ1 and PLCγ2, MAP kinase, Shc and VAV. A primary target molecule iscapable of producing second messenger molecule which is capable offurther amplifying a transduced signal. Second messenger moleculesinclude, but are not limited to diacylglycerol and inositol1,4,5-triphosphate (IP3). Second messenger molecules are capable ofinitiating physiological events which can lead to alterations in genetranscription. For example, production of IP3 can result in release ofintracellular calcium, which can then lead to activation of calmodulinkinase II, which can then lead to serine phosphorylation of a DNAbinding protein referred to as ets-1 proto-onco-protein. Diacylglycerolis capable of activating the signal transduction protein, protein kinaseC which affects the activity of the AP 1 DNA binding protein complex.Signal transduction pathways can lead to transcriptional activation ofgenes such as c-fos, egr-1, and c-myc.

[0100] Shc can be thought of as an adaptor molecule. An adaptor moleculecomprises a protein that enables two other proteins to form a complex(e.g., a three molecule complex). Shc protein enables a complex to formwhich includes Grb2 and SOS. Shc comprises an SH2 domain that is capableof associating with the SH2 domain of Grb2.

[0101] Molecules of a signal transduction pathway can associate with oneanother using recognition sequences. Recognition sequences enablespecific binding between two molecules. Recognition sequences can varydepending upon the structure of the molecules that are associating withone another. A molecule can have one or more recognition sequences, andas such can associate with one or more different molecules.

[0102] Signal transduction pathways for MIRR complexes are capable ofregulating the biological functions of a cell. Such functions caninclude, but are not limited to the ability of a cell to grow, todifferentiate and to secrete cellular products. MIRR-induced signaltransduction pathways can regulate the biological functions of specifictypes of cells involved in particular responses by an animal, such asimmune responses, inflammatory responses and allergic responses. Cellsinvolved in an immune response can include, for example, B cells, Tcells, macrophages, dendritic cells, natural killer cells and plasmacells. Cells involved in inflammatory responses can include, forexample, basophils, mast cells, eosinophils, neutrophils andmacrophages. Cells involved in allergic responses can include, forexample mast cells, basophils, B cells, T cells and macrophages.

[0103] In exemplary embodiments of the subject assay, the detectionsignal is a second messangers, such as a phosphorylated src-likeprotein, includes reporter constructs which include transcriptionalregulatory elements such as serum response element (SRE),12-O-tetradecanoyl-phorbol-13-acetate response element, cyclic AMPresponse element, c-fos promoter, or a CREB-responsive element.

[0104] VII. Nuclear Receptors.

[0105] In another embodiment, the target receptor is a nuclear receptor.The nuclear receptors may be viewed as ligand-dependent transcriptionfactors. These receptors provide a direct link between extracellularsignals, mainly hormones, and transcriptional responses. Theirtranscriptional activation function is regulated by endogenous smallmolecules, such as steroid hormones, vitamin D, ecdysone, retinoic acidsand thyroid hormones, which pass readily through the plasma membrane andbind their receptors inside the cell (Laudet and Adelmant (1995) CurrentBiology 5:124). The majority of these receptors appear to contain threedomains: a variable amino terminal domain; a highly conserved,DNA-binding domain and a moderately conserved, carboxyl-terminalligand-binding domain (Power et al. (1993) Curr. Opin. Cell Biol.5:499-504). Examples include the estrogen, progesterone, androgen,thyroid hormone and mineralocorticoid receptors. In addition to theknown steroid receptors, at least 40 orphan members of this superfamilyhave been identified. (Laudet et al., (1992) EMBO J. 11:1003-1013).There are at least four groups of orphan nuclear receptors representedby NGF1, FTZ-F1, Rev-erbs, and RARs, which are by evolutionarystandards, only distantly related to each other (Laudet et al. supra).While the steroid hormone receptors bind exclusively as homodimers to apalindrome of their hormone responsive element other nuclear receptorsbind as heterodimers. Interestingly, some orphan receptors bind asmonomers to similar response elements and require for their function aspecific motif that is rich in basic amino-acid residues and is locatedcorboxy-terminal to the DNA-binding domain (Laudet and Adelmant supra.)In preferred embodiments, the subject assay is derived to utilize ahormone-dependent reporter construct for selection. For instance,glucocorticoid response elements (GREs) and thyroid receptorenhancer-like DNA sequences (TREs) can be used to drive expression ofreporter construct in response to hormone binding to hormone receptors.GRE's are enhancer-like DNA sequences that confer glucocorticoidresponsiveness via interaction with the glucocorticoid receptor. SeePayvar, et al. (1983) Cell 35:381 and Schiedereit et al. (1983) Nature304:749. TRE's are similar to GRE's except that they confer thyroidhormone responsiveness via interaction with thyroid hormone receptor.Turning now to the interaction of hormones and receptors, it is knownthat a steroid or thyroid hormone enters cells by facilitated diffusionand binds to its specific receptor protein, initiating an allostericalteration of the protein. As a result of this alteration, thehormone/receptor complex is capable of binding to certain specific siteson transcriptional regulatory sequence with high affinity.

[0106] It is also known that many of the primary effects of steroid andthyroid hormones involve increased transcription of a subset of genes inspecific cell types. Moreover, there is evidence that activation oftranscription (and, consequently, increased expression) of genes whichare responsive to steroid and thyroid hormones (through interaction ofchromatin with hormone receptor/hormone complex) is effected throughbinding of the complex to enhancers associated with the genes.

[0107] In any case, a number of steroid hormone and thyroid hormoneresponsive transcriptional control units, some of which have been shownto include enhancers, have been identified. These include the mousemammary tumor virus 5′-long terminal repeat (MMTV LTR), responsive toglucocorticoid, aldosterone and androgen hormones; the transcriptionalcontrol units for mammalian growth hormone genes, responsive toglucocorticoids, estrogens, and thyroid hormones; the transcriptionalcontrol units for mammalian prolactin genes and progesterone receptorgenes, responsive to estrogens; the transcriptional control units foravian ovalbumin genes, responsive to progesterones; mammalianmetallothionein gene transcriptional control units, responsive toglucocorticoids; and mammalian hepatic alpha 2u -globulin genetranscriptional control units, responsive to androgens, estrogens,thyroid hormones and glucocorticoids. Such steroid hormone and thyroidhormone responsive transcriptional control units can be used to generatereporter constructs which are sensitive to agonists and antagonists ofthe steroid hormone and/or thyroid hormone receptors. See, for example,U.S. Pat. Nos. 5,298,429 and 5,071,773, both to Evans, et. al. Moreover,the art describes the functional expression of such receptors in yeast.See also for example, Caplan et al. (1995) J Biol Chem 270:5251-7; andBaniahmad et al. (1995) Mol Endocrinol 9: 34-43.

[0108] VIII. Receptor Tyrosine Kinases.

[0109] In still another embodiment, the target receptor is a receptortyrosine kinase. The receptor tyrosine kinases can be divided into fivesubgroups on the basis of structural similarities in their extracellulardomains and the organization of the tyrosine kinase catalytic region intheir cytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the eph/eck familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316-6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence (Hanks et al. (1988) Science 241:42-52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

[0110] The family with by far the largest number of known members is theEPH family. Since the description of the prototype, the EPH receptor(Hirai et al. (1987) Science 238:1717-1720), sequences have beenreported for at least ten members of this family, not countingapparently orthologous receptors found in more than one species.Additional partial sequences, and the rate at which new members arestill being reported, suggest the family is even larger (Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J. Neurosci Res 37:129-143; and references in Tuziand Gullick (1994) Br J Cancer 69:417-421). Remarkably, despite thelarge number of members in the EPH family, all of these molecules wereidentified as orphan receptors without known ligands.

[0111] The expression patterns determined for some of the EPH familyreceptors have implied important roles for these molecules in earlyvertebrate development. In particular, the timing and pattern ofexpression of sek, mek4 and some of the other receptors during the phaseof gastrulation and early organogenesis has suggested functions forthese receptors in the important cellular interactions involved inpatterning the embryo at this stage (Gilardi-Hebenstreit et al. (1992)Oncogene 7:2499-2506; Nieto et al. (1992) Development 116:1137-1150;Henkemeyer et al., supra; Ruiz et al., supra; and Xu et al., supra).Sek, for example, shows a notable early expression in the two areas ofthe mouse embryo that show obvious segmentation, namely the somites inthe mesoderm and the rhombomeres of the hindbrain; hence the name sek,for segmentally expressed kinase (Gilardi-Hebenstreit et al., supra;Nieto et al., supra). As in Drosophila, these segmental structures ofthe mammalian embryo are implicated as important elements inestablishing the body plan. The observation that Sek expression precedesthe appearance of morphological segmentation suggests a role for sek informing these segmental structures, or in determining segment-specificcell properties such as lineage compartmentation (Nieto et al., supra).Moreover, EPH receptors have been implicated, by their pattern ofexpression, in the development and maintenance of nearly every tissue inthe embryonic and adult body. For instance, EPH receptors have beendetected throughout the nervous system, the testes, the cartilaginousmodel of the skeleton, tooth primordia, the infundibular component ofthe pituitary, various epithelia tissues, lung, pancreas, liver andkidney tissues. Observations such as this have been indicative ofimportant and unique roles for EPH family kinases in development andphysiology, but further progress in understanding their action has beenseverely limited by the lack of information on their ligands.

[0112] As used herein, the terms “EPH receptor” or “EPH-type receptor”refer to a class of receptor tyrosine kinases, comprising at leasteleven paralogous genes, though many more orthologs exist within thisclass, e.g. homologs from different species. EPH receptors, in general,are a discrete group of receptors related by homology and easilyreconizable, e.g., they are typically characterized by an extracellulardomain containing a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717-1720; Lindberg et al. (1990) Mol Cell Biol10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J Neurosci Res 37:129-143; and references in Tuzi andGullick (1994) Br J Cancer 69:417-421). Exemplary EPH receptors includethe eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1, tyro4, tyro5,tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk, rtk1,rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk and nukreceptors. The term “EPH receptor” refers to the membrane form of thereceptor protein, as well as soluble extracellular fragments whichretain the ability to bind the ligand of the present invention.

[0113] In exemplary embodiments, the detection signal is provided bydetecting phosphorylation of intracellular proteins, e.g., MEKKs, MEKs,or Map kinases, or by the use of reporter constructs which includetranscriptional regulatory elements responsive to c-fos and/or c-jun.Described infra.

[0114] IX. G Protein-Coupled Receptors.

[0115] One family of signal transduction cascades found in eukaryoticcells utilizes heterotrimeric “G proteins.” Many different G proteinsare known to interact with receptors. G protein signaling systemsinclude three components: the receptor itself, a GTP-binding protein (Gprotein), and an intracellular target protein.

[0116] The cell membrane acts as a switchboard. Messages arrivingthrough different receptors can produce a single effect if the receptorsact on the same type of G protein. On the other hand, signals activatinga single receptor can produce more than one effect if the receptor actson different kinds of G proteins, or if the G proteins can act ondifferent effectors.

[0117] In their resting state, the G proteins, which consist of alpha(α), beta (β) and gamma (γ) subunits, are complexed with the nucleotideguanosine diphosphate (GDP) and are in contact with receptors. When ahormone or other first messenger binds to receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant nucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the α subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

[0118] Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the asubunit, several different β and γ structures have been reported. Thereare, additionally, several different G protein-dependent effectors.

[0119] Most G protein-coupled receptors are comprised of a singleprotein chain that is threaded through the plasma membrane seven times.Such receptors are often referred to as seven-transmembrane receptors(STRs). More than a hundred different STRs have been found, includingmany distinct receptors that bind the same ligand, and there are likelymany more STRs awaiting discovery.

[0120] In addition, STRs have been identified for which the naturalligands are unknown; these receptors are termed “orphan” Gprotein-coupled receptors, as described above. Examples includereceptors cloned by Neote et al. (1993) Cell 72, 415; Kouba et al. FEBSLett. (1993) 321, 173; Birkenbach et al.(1993) J. Virol. 67, 2209.

[0121] The “exogenous receptors” of the present invention may be any Gprotein-coupled receptor which is exogenous to the cell which is to begenetically engineered for the purpose of the present invention. Thisreceptor may be a plant or animal cell receptor. Screening for bindingto plant cell receptors may be useful in the development of, e.g.,herbicides. In the case of an animal receptor, it may be of invertebrateor vertebrate origin. If an invertebrate receptor, an insect receptor ispreferred, and would facilitate development of insecticides. Thereceptor may also be a vertebrate, more preferably a mammalian, stillmore preferably a human, receptor. The exogenous receptor is alsopreferably a seven transmembrane segment receptor.

[0122] Known ligands for G protein coupled receptors include: purinesand nucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin andthe like; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, adrenaline,adrenaline., histamine, noradrenaline, noradrenaline, noradrenaline,tyramine/octopamine and other related compounds; peptides such asadrenocorticotrophic hormone (acth), melanocyte stimulating hormone(msh), melanocortins, neurotensin (nt), bombesin and related peptides,endothelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebratetachykinin-like peptides, substance k (nk2), substance p (nk1),neuropeptide y (npy), thyrotropin releasing-factor (trf), bradykinin,angiotensin ii, beta-endorphin, c5a anaphalatoxin, calcitonin,chemokines (also called intercrines), corticotrophic releasing factor(crf), dynorphin, endorphin, fmlp and other formylated peptides,follitropin (fsh), fungal mating pheremones, galanin, gastric inhibitorypolypeptide receptor (gip), glucagon-like peptides (glps), glucagon,gonadotropin releasing hormone (gnrh), growth hormone releasinghormone(ghrh), insect diuretic hormone, interleukin-8, leutropin(lh/hcg), met-enkephalin, opioid peptides, oxytocin, parathyroid hormone(pth) and pthrp, pituitary adenylyl cyclase activiating peptide (pacap),secretin, somatostatin, thrombin, thyrotropin (tsh), vasoactiveintestinal peptide (vip), vasopressin, vasotocin; eicosanoids such asip-prostacyclin, pg-prostaglandins, tx-thromboxanes; retinal basedcompounds such as vertebrate 11-cis retinal, invertebrate 11-cis retinaland other related compounds; lipids and lipid-based compounds such ascannabinoids, anandamide, lysophosphatidic acid, platelet activatingfactor, leukotrienes and the like; excitatory amino acids and ions suchas calcium ions and glutamate.

[0123] Suitable examples of G-protein coupled receptors include, but arenot limited to, dopaminergic, muscarinic cholinergic, a-adrenergic,b-adrenergic, opioid (including delta and mu), cannabinoid,serotoninergic, and GABAergic receptors. Preferred receptors include the5HT family of receptors, dopamine receptors, C5a receptor and FPRL-1receptor, cyclo-histidyl-proline-diketoplperazine receptors, melanocytestimulating hormone release inhibiting factor receptor, and receptorsfor neurotensin, thyrotropin releasing hormone, calcitonin,cholecytokinin-A, neurokinin-2, histamine-3, cannabinoid, melanocortin,or adrenomodulin, neuropeptide-Y1 or galanin. Other suitable receptorsare listed in the art. The term “receptor,” as used herein, encompassesboth naturally occurring and mutant receptors.

[0124] Many of these G protein-coupled receptors, like the yeast a- andα-factor receptors, contain seven hydrophobic amino acid-rich regionswhich are assumed to lie within the plasma membrane. Specific human Gprotein-coupled STRs for which genes have been isolated and for whichexpression vectors could be constructed include those listed herein andothers known in the art. Thus, the gene would be operably linked to apromoter functional in the cell to be engineered and to a signalsequence that also functions in the cell. For example in the case ofyeast, suitable promoters include Ste2, Ste3 and ga110. Suitable signalsequences include those of Ste2, Ste3 and of other genes which encodeproteins secreted by yeast cells. Preferably, when a yeast cell is used,the codons of the gene would be optimized for expression in yeast. SeeHoekema et al., (1987) Mol. Cell. Biol., 7:2914-24; Sharp, et al.,(1986)14:5125-43.

[0125] The homology of STRs is discussed in Dohlman et al., Ann. Rev.Biochem., (1991) 60:653-88. When STRs are compared, a distinct spatialpattern of homology is discernible. The transmembrane domains are oftenthe most similar, whereas the N- and C-terminal regions, and thecytoplasmic loop connecting transmembrane segments V and VI are moredivergent.

[0126] The functional significance of different STR regions has beenstudied by introducing point mutations (both substitutions anddeletions) and by constructing chimeras of different but related STRs.Synthetic peptides corresponding to individual segments have also beentested for activity. Affinity labeling has been used to identify ligandbinding sites.

[0127] It is conceivable that a foreign receptor which is expressed inyeast will functionally integrate into the yeast membrane, and thereinteract with the endogenous yeast G protein. More likely, either thereceptor will need to be modified (e.g., by replacing its V-VI loop withthat of the yeast STE2 or STE3 receptor), or a compatible G proteinshould be provided.

[0128] If the wild-type exogenous G protein-coupled receptor cannot bemade functional in yeast, it may be mutated for this purpose. Acomparison would be made of the amino acid sequences of the exogenousreceptor and of the yeast receptors, and regions of high and lowhomology identified. Trial mutations would then be made to distinguishregions involved in ligand or G protein binding, from those necessaryfor functional integration in the membrane. The exogenous receptor wouldthen be mutated in the latter region to more closely resemble the yeastreceptor, until functional integration was achieved. If this wereinsufficient to achieve functionality, mutations would next be made inthe regions involved in G protein binding. Mutations would be made inregions involved in ligand binding only as a last resort, and then aneffort would be made to preserve ligand binding by making conservativesubstitutions whenever possible.

[0129] Preferably, the yeast genome is modified so that it is unable toproduce the yeast receptors which are homologous to the exogenousreceptors in functional form. Otherwise, a positive assay score mightreflect the ability of a peptide to activate the endogenous Gprotein-coupled receptor, and not the receptor of interest.

[0130] A. Chemoattractant Receptors

[0131] The N-formyl peptide receptor is a classic example of a calciummobilizing G protein-coupled receptor expressed by neutrophils and otherphagocytic cells of the mammalian immune system (Snyderman et al. (1988)In Inflammation: Basic Principles and Clinical Correlates, pp. 309-323).N-formyl peptides of bacterial origin bind to the receptor and engage acomplex activation program that results in directed cell movement,release of inflammatory granule contents, and activation of a latentNADPH oxidase which is important for the production of metabolites ofmolecular oxygen. This pathway initiated by receptor-ligand interactionis critical in host protection from pyogenic infections. Similar signaltransduction occurs in response to the inflammatory peptides C5a andIL-8.

[0132] Two other formyl peptide receptor like (FPRL) genes have beencloned based on their ability to hybridize to a fragment of the NFPRcDNA coding sequence. These have been named FPRL1 (Murphy et al. (1992)J. Biol Chem. 267:7637-7643) and FPRL2 (Ye et al. (1992) Biochem BiophysRes. Comm. 184:582-589). FPRL2 was found to mediate calcium mobilizationin mouse fibroblasts transfected with the gene and exposed to formylpeptide. In contrast, although FPRL1 was found to be 69% identical inamino acid sequence to NFPR, it did not bind prototype N-formyl peptidesligands when expressed in heterologous cell types. This lead to thehypothesis of the existence of an as yet unidentified ligand for theFPRL 1 orphan receptor (Murphy et al. supra).

[0133] Using the technology described herein a ligand has been clonedfor these orphan receptors.

[0134] B. G Proteins

[0135] In the case of an exogenous G-protein coupled receptor, the yeastcell must be able to produce a G protein which is activated by theexogenous receptor, and which can in turn activate the yeasteffector(s). The art suggests that the endogenous yeast Gα subunit(e.g., GPA) will be often be sufficiently homologous to the “cognate” Gαsubunit which is natively associated with the exogenous receptor forcoupling to occur. More likely, it will be necessary to geneticallyengineer the yeast cell to produce a foreign Gα subunit which canproperly interact with the exogenous receptor. For example, the Gαsubunit of the yeast G protein may be replaced by the Gα subunitnatively associated with the exogenous receptor.

[0136] Dietzel and Kurjan, (1987) Cell, 50:1001) demonstrated that ratGαs functionally coupled to the yeast Gβγ complex. However, rat Gαi2complemented only when substantially overexpressed, while Gα0 did notcomplement at all. Kang, et al., Mol. Cell. Biol., (1990)10:2582).Consequently, with some foreign Gα subunits, it is not feasible tosimply replace the yeast Gα.

[0137] If the exogenous G protein coupled receptor is not adequatelycoupled to yeast Gβγ by the Gα subunit natively associated with thereceptor, the Gα subunit may be modified to improve coupling. Thesemodifications often will take the form of mutations which increase theresemblance of the Gα subunit to the yeast Gα while decreasing itsresemblance to the receptor-associated Gα. For example, a residue may bechanged so as to become identical to the corresponding yeast Gα residue,or to at least belong to the same exchange group of that residue. Aftermodification, the modified Gα subunit might or might not be“substantially homologous” to the foreign and/or the yeast Gα subunit.

[0138] The modifications are preferably concentrated in regions of theGα which are likely to be involved in Gβγ binding. In some embodiments,the modifications will take the form of replacing one or more segmentsof the receptor-associated Gα with the corresponding yeast Gαsegment(s), thereby forming a chimeric Gα subunit. (For the purpose ofthe appended claims, the term “segment” refers to three or moreconsecutive amino acids.) In other embodiments, point mutations may besufficient.

[0139] This chimeric Gα subunit will interact with the exogenousreceptor and the yeast Gβγ complex, thereby permitting signaltransduction. While use of the endogenous yeast Gβγ is preferred, if aforeign or chimeric Gβγ is capable of transducing the signal to theyeast effector, it may be used instead.

[0140] C. Gα Structure

[0141] Some aspects of Gα structure are relevant to the design ofmodified Gα subunits. The amino terminal 66 residues of GPA1 are alignedwith the cognate domains of human Gαs, Gαi2, Gαi3, Gα16 and transducin.In the GPA41Gα hybrids, the amino terminal 41 residues (derived fromGPA1) are identical, end with the sequence-LEKQRDKNE- and are underlinedfor emphasis. All residues following the glutamate (E) residue atposition 41 are contributed by the human Gα subunits, including theconsensus nucleotide binding motif -GxGxxG-. Periods in the sequencesindicate gaps that have been introduced to maximize alignments in thisregion. Codon bias is mammalian. For alignments of the entire codingregions of GPA1 with Gαs, Gβi, and GαO, Gαq and Gαz, see Dietzel andKurjan (1987, Cell 50:573) and Lambright, et al. (1994, Nature369:621-628). Additional sequence information is provided by Mattera, etal. (1986, FEBS Lett 206:36-41), Bray, et al. (1986, Proc. Natl. Acad.Sci USA 83:8893-8897) and Bray, et al. (1987, Proc Natl. Acad Sci USA84:5115-5119).

[0142] The gene encoding a G protein homolog of S. cerevisiae was clonedindependently by Dietzel and Kurjan (supra) (SCG1) and by Nakafuku, etal. (1987 Proc Natl Acad Sci 84:2140-2144) (GPA1). Sequence analysisrevealed a high degree of homology between the protein encoded by thisgene and mammalian Gα. GPA1 encodes a protein of 472 amino acids, ascompared with approximately 340-350 a.a. for most mammalian Gα subunitsin four described families, Gαs, Gαi, Gαq and Gα12/13. Nevertheless,GPA1 shares overall sequence and structural homology with all Gαproteins identified to date. The highest overall homology in GPA1 is tothe Gαi family (48% identity, or 65% with conservative substitutions)and the lowest is to GQS (33% identity, or 51% with conservativesubstitutions) (Nakafuku, et al., supra).

[0143] The regions of high sequence homology among Gα subunits aredispersed throughout their primary sequences, with the regions sharingthe highest degree of homology mapping to sequence that comprises theguanine nucleotide binding/GTPase domain. This domain is structurallysimilar to the aβ fold of ras proteins and the protein synthesiselongation factor EF-Tu. This highly conserved guaninenucleotide-binding domain consists of a six-stranded β sheet surroundedby a set of five α-helices. It is within these β sheets and α helicesthat the highest degree of conservation is observed among all Gαproteins, including GPA1. The least sequence and structural homology isfound in the intervening loops between the β sheets and α helices thatdefine the core GTPase domain. There are a total of four “interveningloops” or “inserts” present in all Gα subunits. In the crystalstructures reported to date for the GDP- and GTPγS-liganded forms ofbovine rod transducin (Noel, et al. (1993) Nature 366:654-663);(Lambright, et al. (1994) Nature 369:621-628), the loop residues arefound to be outside the core GTPase structure. Functional roles forthese loop structures have been established in only a few instances. Adirect role in coupling to phosphodiesterase-γ has been demonstrated forresidues within inserts 3 and 4 of Gαt (Rarick, et al. (1992) Science256:1031-1033); (Artemyev, et al. (1992) J. Biol. Chem.267:25067-25072), while a “GAP-like” activity has been ascribed to thelargely α-helical insert 1 domain of GαS (Markby, et al. (1993) Science262:1805-1901).

[0144] While the amino- and carboxy-termini of Gα subunits do not sharestriking homology either at the primary, secondary, or tertiary levels,there are several generalizations that can be made about them. First,the amino termini of Gα subunits have been implicated in the associationof Gα with Gβγ complexes and in membrane association via N-terminalmyristoylation. In addition, the carboxy-termini have been implicated inthe association of Gαβγ heterotrimeric complexes with G protein-coupledreceptors (Sullivan, et al. (1987) Nature 330:758-760); West, et al.(1985) J. Biol. Chem. 260:14428-14430); (Conklin, et al. (1993) Nature363:274-276). Data in support of these generalizations about thefunction of the N-terminus derive from several sources, including bothbiochemical and genetic studies.

[0145] As indicated above, there is little if any sequence homologyshared among the amino termini of Gα subunits. The amino terminaldomains of Gα subunits that precede the first β-sheet (containing thesequence motif -LLLLGAGESG-; see Noel, et al. (supra) for the numberingof the structural elements of Gα subunits) vary in length from 41 aminoacids (GPA1) to 31 amino acids (Gαt). Most Gα subunits share theconsensus sequence for the addition of myristic acid at their aminotermini (MGXaaS-), although not all Gα subunits that contain this motifhave myristic acid covalently associated with the glycine at position 2(Speigel, et al. (1991) TIBS 16:338-3441). The role of thispost-translational modification has been inferred from studies in whichthe activity of mutant Gα subunits from which the consensus sequence formyristoylation has been added or deleted has been assayed (Mumby et al.(1990) Proc. Natl. Acad. Sci. USA 87: 728-732; (Linder, et al. (1991) J.Biol Chem. 266:4654-4659); Gallego, et al. (1992) Proc. Natl. Acad. Sci.USA 89:9695-9699). These studies suggest two roles for N-terminalmyristoylation. First, the presence of amino-terminal myristic acid hasin some cases been shown to be required for association of Gα subunitswith the membrane, and second, this modification has been demonstratedto play a role in modulating the association of Gα subunits with Gβγcomplexes. The role of myristoylation of the GPA1 gene products, atpresent, unknown.

[0146] In other biochemical studies aimed at examining the role of theamino-terminus of Gα in driving the association between Gα and Gβγsubunits, proteolytically or genetically truncated versions of Gαsubunits were assayed for their ability to associate with Gβγ complexes,bind guanine nucleotides and/or to activate effector molecules. In allcases, Gα subunits with truncated amino termini were deficient in allthree functions (Graf, et al. (1992) J. Biol. Chem. 267:24307-24314);(Journot, et al. (1990) J. Biol. Chem. 265:9009-9015); and (Neer, et al.(1988) J. Biol. Chem 263:8996-9000). Slepak, et al. (1993, J. Biol.Chem. 268:1414-1423) reported a mutational analysis of the N-terminal 56a.a. of mammalian Gαo expressed in Escherichia coli. Molecules with anapparent reduced ability to interact with exogenously added mammalianGβγ were identified in the mutant library. As the authors pointed out,however, the assay used to screen the mutants the extent ofADP-ribosylation of the mutant Gα by pertussis toxin was not acompletely satisfactory probe of interactions between Gα and Gβγ.Mutations identified as inhibiting the interaction of the subunits,using this assay, may still permit the complexing of Gα and Gβγ whilesterically hindering the ribosylation of Gα by toxin. Genetic studiesexamined the role of amino-terminal determinants of Gα in heterotrimersubunit association have been carried out in both yeast systems usingGPA1-mammalian Gα hybrids (Kang, et al. (1990) Mol. Cell. Biol.10:2582-2590) and in mammalian systems using Gαi/Gαs hybrids (Russelland Johnson (1993) Mol. Pharmacol. 44:255-263). In the former studies,gene fusions, composed of yeast GPA1 and mammalian Gα sequences wereconstructed by Kang, et al. (supra) and assayed for their ability tocomplement a gpal null phenotype (i.e., constitutive activation of thepheromone response pathway) in S. cerevisiae. Kang, et al. demonstratedthat wild type mammalian Gαs, Gαi but not Gαo proteins are competent toassociate with yeast Gα and suppress the gpal null phenotype, but onlywhen overexpressed. Fusion proteins containing the amino-terminal 330residues of GPA1 sequence linked to 160, 143, or 142 residues of themammalian Gαs, Gαi and Gαo carboxyl-terminal regions, respectively, alsocoupled to the yeast mating response pathway when overexpressed on highcopy plasmids with strong inducible (CUP) or constitutive (PGK)promoters. All three of these hybrid molecules were able to complementthe gpal null mutation in a growth arrest assay, and were additionallyable to inhibit αfactor responsiveness and mating in tester strains.These last two observations argue that hybrid yeast-mammalian Gαsubunits are capable of interacting directly with yeast Gβγ, therebydisrupting the normal function of the yeast heterotrimer. Fusionscontaining the amino terminal domain of Gαs, Gαi or Gαo, however, didnot complement the gpal null phenotype, indicating a requirement fordeterminants in the amino terminal 330 amino acid residues of GPA1 forassociation and sequestration of yeast Gβγ complexes. Taken together,these data suggest that determinants in the amino terminal region of Gαsubunits determine not only the ability to associate with Gβγ subunitsin general, but also with specific Gβγ subunits in a species-restrictedmanner.

[0147] Hybrid Gαi/Gαs subunits have been assayed in mammalian expressionsystems (Russell and Johnson (supra). In these studies, a large numberof chimeric Gα subunits were assayed for an ability to activate adenylylcyclase, and therefore, indirectly, for an ability to interact with Gβγ(i.e., coupling of Gα to Gβγ=inactive cyclase; uncoupling of Gα fromGβγ=active cyclase). From these studies a complex picture emerged inwhich determinants in the region between residues 25 and 96 of thehybrids were found to determine the state of activation of these allelesas reflected in their rates of guanine nucleotide exchange and GTPhydrolysis and the extent to which they activated adenylyl cyclase invivo. These data could be interpreted to support the hypothesis thatstructural elements in the region between the amino terminal methionineand the ˜1 sheet identified in the crystal structure of Gαt (see Noel,et al. supra and Lambright, et al. supra) are involved in determiningthe state of activity of the heterotrimer by (1) drivingassociation/dissociation between Gα and Gβγ subunits; (2) drivingGDP/GTP exchange. While there is no direct evidence provided by thesestudies to support the idea that residues in this region of Gα andresidues in Gβγ subunits contact one another, the data nonethelessprovide a positive indication for the construction of hybrid Gα subunitsthat retain function. There is, however, a negative indicator thatderives from this work in that some hybrid constructs resulted inconstitutive activation of the chimeric proteins (i.e., a loss ofreceptor-dependent stimulation of Gβγ dissociation and effectoractivation).

[0148] D. Construction of Chimeric Gα Subunits.

[0149] In designing Gα subunits capable of transmitting, in yeast,signals originating at mammalian G protein-coupled receptors, twogeneral desiderata were recognized. First, the subunits should retain asmuch of the sequence of the native mammalian proteins as possible.Second, the level of expression for the heterologous components shouldapproach, as closely as possible, the level of their endogenouscounterparts. The results described by King, et al. (1990, Science250:121-123) for expression of the human β2-adrenergic receptor and Gasin yeast, taken together with negative results obtained by Kang, et al.(supra) with full-length mammalian Gα subunits other than Gαs, led us tothe following preferences for the development of yeast strains in whichmammalian G protein-coupled receptors could be linked to the pheromoneresponse pathway.

[0150] 1. Mammalian Gα subunits will be expressed using the nativesequence of each subunit or, alternatively, as minimal gene fusions withsequences from the amino-terminus of GPA1 replacing the homologousresidues from the mammalian Gα subunits.

[0151] 2. Mammalian Gα subunits will be expressed from the GPA1 promotoreither on low copy plasmids or after integration into the yeast genomeas a single copy gene.

[0152] 3. Endogenous Gβγ subunits will be provided by the yeast STE4 andSTE18 loci.

[0153] E. Site-Directed Mutagenesis Versus Random Mutagenesis

[0154] There are two general approaches to solving structure-functionproblems of the sort presented by attempts to define the determinantsinvolved in mediating the association of the subunits that comprise theG protein heterotrimer. The first approach, discussed above with respectto hybrid constructs, is a rational one in which specific mutations oralterations are introduced into a molecule based upon the availableexperimental evidence. In a second approach, random mutagenesistechniques, coupled with selection or screening systems, are used tointroduce large numbers of mutations into a molecule, and thatcollection of randomly mutated molecules is then subjected to aselection for the desired phenotype or a screen, in which the desiredphenotype can be observed against a background of undesirablephenotypes. With random mutagenesis one can mutagenize an entiremolecule or one can proceed by cassette mutagenesis. In the formerinstance, the entire coding region of a molecule is mutagenized by oneof several methods (chemical, PCR, doped oligonucleotide synthesis) andthat collection of randomly mutated molecules is subjected to selectionor screening procedures. Random mutagenesis can be applied in this wayin cases where the molecule being studied is relatively small and thereare powerful and stringent selections or screens available todiscriminate between the different classes of mutant phenotypes thatwill inevitably arise. In the second approach, discrete regions of aprotein, corresponding either to defined structural (i.e. α-helices,β-sheets, turns, surface loops) or functional determinants (e.g.,catalytic clefts, binding determinants, transmembrane segments) aresubjected to saturating or semi-random mutagenesis and these mutagenizedcassettes are re-introduced into the context of the otherwise wild typeallele. Cassette mutagenesis is most useful when there is experimentalevidence available to suggest a particular function for a region of amolecule and there is a powerful selection and/or screening approachavailable to discriminate between interesting and uninteresting mutants.Cassette mutagenesis is also useful when the parent molecule iscomparatively large and the desire is to map the functional domains of amolecule by mutagenizing the molecule in a step-wise fashion, i.e.mutating one linear cassette of residues at a time and then assaying forfunction.

[0155] The present invention contemplates applying random mutagenesis inorder to further delineate the determinants involved in Gα-Gβγassociation. Random mutagenesis may be accomplished by many means,including:

[0156] 1. PCR mutagenesis, in which the error prone Taq polymerase isexploited to generate mutant alleles of Gα subunits, which are assayeddirectly in yeast for an ability to couple to yeast Gβγ.

[0157] 2. Chemical mutagenesis, in which expression cassettes encodingGα subunits are exposed to mutagens and the protein products of themutant sequences are assayed directly in yeast for an ability to coupleto yeast Gβγ.

[0158] 3. Doped synthesis of oligonucleotides encoding portions of theGα gene.

[0159] 4. In vivo mutagenesis, in which random mutations are introducedinto the coding region of Gα subunits by passage through a mutatorstrain of E. coli, XL 1-Red (mutD5 mutS mutT) (Stratagene, Menasa,Wis.).

[0160] The random mutagenesis may be focused on regions suspected to beinvolved in Gα-Gβγ association as discussed in the next section. Randommutagenesis approaches are feasible for two reasons. First, in yeast onehas the ability to construct stringent screens and facile selections(growth vs. death, transcription vs. lack of transcription) that are notreadily available in mammalian systems. Second, when using yeast it ispossible to screen efficiently through thousands of transformantsrapidly. Cassette mutagenesis is immediately suggested by theobservation (see infra) that the GPA₄₁ hybrids couple to the pheromoneresponse pathway. This relatively small region of Gα subunits representsa reasonable target for this type of mutagenesis. Another region thatmay be amenable to cassette mutagenesis is that defining the surface ofthe switch region of Gα subunits that is solvent-exposed in the crystalstructures of Gαi and transducin. From the data described below, thissurface may contain residues that are in direct contact with yeast Gβγsubunits, and may therefore be a reasonable target for mutagenesis.

[0161] F. Rational Design of Chimeric Gα Subunits

[0162] Several classes of rationally designed GPA1-mammalian Gα hybridsubunits have been tested for the ability to couple to yeast βγ. Thefirst, and largest, class of hybrids are those that encode differentlengths of the GPA1 amino terminal domain in place of the homologousregions of the mammalian Gα subunits. This class of hybrid moleculesincludes GPA_(BAMH1), GPA₄₁, GPA_(ID), and GPA_(LW) hybrids, describedbelow. The rationale for constructing these hybrid Gα proteins is basedon results, described above, that bear on the importance of the aminoterminal residues of Gα in mediating interaction with Gβγ.

[0163] Preferably, the yeast Gα subunit is replaced by a chimeric Gαsubunit in which a portion, e.g., at least about 20, more preferably atleast about 40, amino acids, which is substantially homologous with thecorresponding residues of the amino terminus of the yeast Gα, is fusedto a sequence substantially homologous with the main body of a mammalian(or other exogenous) Gα. While 40 amino acids is the suggested startingpoint, shorter or longer portions may be tested to determine the minimumlength required for coupling to yeast Gβγ and the maximum lengthcompatible with retention of coupling to the exogenous receptor. It ispresently believed that only the final 10 or 20 amino acids at thecarboxy terminus of the Gα subunit are required for interaction with thereceptor.

[0164] GPA_(BAMH1) Hybrids.

[0165] Kang et al. supra. described hybrid Gα subunits encoding theamino terminal 310 residues of GPA1 fused to the carboxyl terminal 160,143 and 142 residues, respectively, of GαS, Gαi2, and Gαo. In all casesexamined by Kang et al., the hybrid proteins were able to complement thegrowth arrest phenotype of gpal strains. We have confirmed thesefindings and, in addition, have constructed and tested hybrids betweenGPA1 and Gαi3, Gαq and Gα16. All hybrids of this type that have beentested functionally complement the growth arrest phenotype of gpalstrains.

[0166] GPA41 Hybrids.

[0167] The rationale for constructing a minimal hybrid encoding only 41amino acids of GPA1 relies upon the biochemical evidence for the role ofthe amino-terminus of Gα subunits discussed above, together with thefollowing observation. Gβ and Gγ subunits are known to interact viaα-helical domains at their respective amino-termini (Pronin, et al.(1992) Proc. Natl. Acad. Sci. USA 89:6220-6224); Garritsen, et al.1993). The suggestion that the amino termini of Gα subunits may form anhelical coil and that this helical coil may be involved in associationof Gα with Gβγ (Masters et al (1986) Protein Engineering 1:47-54); Lupaset al.(1992) FEBS Lett. 314:105-108) leads to the hypothesis that thethree subunits of the G-protein heterotrimer interact with one anotherreversibly through the winding and unwinding of their amino-terminalhelical regions. A mechanism of this type has been suggested, as well,from an analysis of leucine zipper mutants of the GCN4 transcriptionfactor (Harbury, et al. (1993) Science 262:1401-1407). The rationale forconstructing hybrids like those described by Kang, et al. supra., thatcontain a majority of yeast sequence and only minimal mammaliansequence, derives from their ability to function in assays of couplingbetween Gα and Gβγ subunits. However, these chimeras had never beenassayed for an ability to couple to both mammalian G protein-coupledreceptors and yeast Gβγ subunits, and hence to reconstitute a hybridsignaling pathway in yeast.

[0168] GPA₄₁ hybrids that have been constructed and tested include Gαs,Gαi2, Gαi3, Gαq, Gαo_(a), Gαo_(b) and Gα16. Hybrids of Gαs, Gαi2, Gαi3,and Gα16 functionally complement the growth arrest phenotype of gpalstrains, while GPA₄₁ hybrids of Gαo_(a) and Gαo_(b) do not. In additionto being tested in a growth arrest assay, these constructs have beenassayed in the more sensitive transcriptional assay for activation of afus1p-HIS3 gene. In both of these assays, the GPA₄₁-Gαs hybrid couplesless well than the GPα₄₁-i2, -i3, and -16 hybrids, while theGPα₄₁-o_(a), and -o_(b) hyrids do not function in either assay.

[0169] Several predictive algorithms indicate that the amino terminaldomain up to the highly conserved sequence motif-LLLLGAGESG- (the firstL in this motif is residue 43 in GPA1) forms a helical structure withamphipathic character. Assuming that a heptahelical repeat unit, thefollowing hybrids between GPA1 and GαS can be used to define the numberof helical repeats in this motif necessary for hybrid function:

[0170] GPA1-7/Gαs8-394

[0171] GPA1-14/Gαs15-394

[0172] GPA1-21/Gαs22-394

[0173] GPA1-28/Gαs29-394

[0174] GPA1-351Gαs36-394

[0175] GPA1-42/Gαs43-394

[0176] In these hybrids, the prediction is that the structural repeatunit in the amino terminal domain up to the tetra-leucine motif is 7,and that swapping sequences in units of 7 will in effect amount to aswap of unit turns of turns of the helical structure that comprises thisdomain.

[0177] A second group of “double crossover’” hybrids of this class arethose that are aligned on the first putative heptad repeat beginningwith residue G11 in GPA1. In these hybrids, helical repeats are swappedfrom GPA1 into a GaS backbone one heptad repeat unit at a time.

[0178] GαS1-10/GPA11-17/Gαs18-394

[0179] GαS1-17/GPA18-24/GαS25-394

[0180] GαS1-17/GPA25-31/GαS32-394

[0181] GαS?-17/GPA32-38/GαS39-394

[0182] The gap that is introduced between residues 9 and 10 in the GaSsequence is to preserve the alignment of the -LLLLGAGE-sequence motif.This class of hybrids can be complemented by cassette mutagenesis ofeach heptad repeat followed by screening of these collections of“heptad” libraries in standard coupling assays.

[0183] A third class of hybrids based on the prediction that the aminoterminus forms a helical domain with a heptahelical repeat unit arethose that effect the overall hydrophobic or hydrophilic character ofthe opposing sides of the predicted helical structure (See Lupas et al.supra). In this model, the a and d positions of the heptad repeatabcdefg are found to be conserved hydrophobic residues that define oneface of the helix, while the e and g positions define the charged faceof the helix. In this class of hybrids, the sequence of the GaS parentis maintained except for specific substitutions at one or more of thefollowing critical residues to render the different helical faces of GaSmore “GPA1-like”

[0184] K8Q

[0185] +I-10

[0186] E1OG

[0187] Q12E

[0188] R13S

[0189] N14D

[0190] E15P

[0191] E15F

[0192] K17L

[0193] E21R

[0194] K28Q

[0195] K32L

[0196] V36R

[0197] This collection of single mutations could be screened forcoupling efficiency to yeast Gβγ and then constructed in combinations(double and greater if necessary).

[0198] A fourth class of hybrid molecules that span this region ofGPA1-Gα hybrids are those that have junctions between GPA1 and Gαsubunits introduced by three primer PCR. In this approach, the twooutside primers are encoded by sequences at the initiator methionine ofGPA1 on the 5′ side and at the tetraleucine motif of GαS (for example)on the 3′ side. A series of junctional primers spanning differentjunctional points can be mixed with the outside primers to make a seriesof molecules each with different amounts of GPA1 and GαS sequences,respectively.

[0199] GPA_(ID) and GPA_(LW) Hybrids.

[0200] The regions of high homology among Gβγ subunits that have beenidentified by sequence alignment are interspersed throughout themolecule. The G1 region containing the highly conserved -GSGESGDST-motifis followed immediately by a region of very low sequence consevation,the “il” or insert 1 region. Both sequence and length vary considerablyamong the il regions of the Gα subunits. By aligning the sequences of Gαsubunits, the conserved regions bounding the il region were identifiedand two additional classes of GPA1-Gα hybrids were constructed. TheGPAID hybrids encode the amino terminal 102 residues of GPA1 (up to thesequence -QARKLGIQ-) fused in frame to mammalian Gα subunits, while theGPALW hybrids encode the amino terminal 244 residues of GPA1 (up to thesequence LIHEDIAKA- in GPA1). The reason for constructing the GPA_(ID)and GPA_(LW) hybrids was to test the hypothesis that the il region ofGPA1 is required for mediating the interaction of GPA1 with yeast Gβγsubunits, for the stable expression of the hybrid molecules, or forfunction of the hybrid molecules. The GPA_(ID) hybrids contain the aminoterminal domain of GPA1 fused to the il domain of mammalian subunits,and therefore do not contain the GPA1 il region, while the GPA_(LW)hybrids contain the amino terminal 244 residues of GPA1 including theentire il region (as defined by sequence alignments). Hybrids of bothGPA_(ID) and GPA_(LW) classes were constructed for GαS, C-αi2, Gαi3,Gαo_(a), and Gα16; none of these hybrids complemented the gpal growtharrest phenotype.

[0201] Subsequent to the construction and testing of the GPA_(ID) andGPA_(LW) classes of hybrids, the crystal structures of G_(transducin) inboth the GDP and GTPγS-liganded form, and the crystal structure ofseveral Gαil variants in the GTPγS-liganded and GDP-AlF₄ forms werereported (Noel et al. supra; Lambright et al. supra; and Coleman etal.(1994) Science 265:1405-1412). The crystal structures reveal that theilregion defined by sequence alignment has a conserved structure that iscomprised of six alpha helices in a rigid array, and that the junctionschosen for the construction of the GPA_(ID) and GPA_(LW) hybrids werenot compatible with conservation of the structural features of the ilregion observed in the crystals. The junction chosen for the GPA_(ID)hybrids falls in the center of the long αA helix; chimerization of thishelix in all likelihood destabilizes it and the protein structure ingeneral. The same is true of the junction chosen for the GPALW hybridsin which the crossover point between GPA1 and the mammalian Gα subunitfalls at the end of the short αC helix and therefore may distort it anddestabilize the protein.

[0202] The failure of the GPA_(ID) and GPA_(LW) hybrids is predicted tobe due to disruption of critical structural elements in the il region asdiscussed above. Based upon new alignments and the data presented inNoel et al (supra), Lambright et al (supra), and Coleman et al (supra),this problem can be averted with the ras-like core domain and the ilhelical domain are introduced outside of known structural elements likealpha-helices.

[0203] Hybrid A GαS1-67/GPA66-299/GαS203-394

[0204] This hybrid contains the entire il insert of GPA1 interposed intothe GαS sequence.

[0205] Hybrid B GPA1-41/GαS4443-67/GPA66-299/GαS203-394

[0206] This hybrid contains the amino terminal 41 residues of GPA1 inplace of the 42 amino terminal residues of GαS found in Hybrid A.

[0207] Gαs Hybrids.

[0208] There is evidence that the “switch region” encoded by residues171-237 of Gα transducin (using the numbering of (Noel et al (supra)also plays a role in Gβγ coupling. First, the G226A mutation in GαSprevents the GTP-induced conformational change that occurs with exchangeof GDP for GTP upon receptor activation by ligand. This residue maps tothe highly conserved sequence -DVGGQ-, present in all Gα subunits and isinvolved in GTP hydrolysis. In both the Gαt and Gα il crystalstructures, this sequence motif resides in the loop that connects the β3sheet and the α2 helix in the guanine nucleotide binding core. Inaddition to blocking the conformational change that occurs upon GTPbinding, this mutation also prevents dissociation of GTP-liganded Gαsfrom Gβγ. Second, crosslinking data reveals that a highly conservedcysteine residue in the α2 helix (C215 in Gαo, C210 in Gαt) can becrosslinked to the carboxy terminal region of Gβ subunits. Finally,genetic evidence (Whiteway et al. (1993) Mol Cell Biol. 14:3233-3239)identifies an important single residue in GPA1 (E307) in the β2 sheet ofthe core structure that may be in direct contact with βγ. A mutation inthe GPA1 protein at this position suppresses the constitutive signallingphenotype of a variety of STE4 (Gβ) dominant negative mutations that arealso known to be defective in Gα-Gβγ association (as assessed intwo-hybrid assay in yeast as well as by more conventional genetictests).

[0209] We have tested the hypothesis that there are switch regiondeterminants involved in the association of Gα with Gβγ by constructinga series of hybrid Gα proteins encoding portions of GPA1 and GαS indifferent combinations.

[0210] Two conclusions may be drawn. First, in the context of the aminoterminus of GαS, the GPA1 switch region suppresses coupling to yeast Gβγ(SGS), while in the context of the GPA1 amino terminus the GPA1 switchregion stabilizes coupling with Gβγ (Gβγ-SGS). This suggests that thesetwo regions of GPA1 collaborate to allow interactions between Gαsubunits and Gβγ subunits. This conclusion is somewhat mitigated by theobservation that the GPA₄₁-Gαs hybrid that does not contain the GPA1switch region is able to complement the growth arrest phenotype of gpalstrains. We have not to date noted a quantitative difference between thebehavior of the GPA₄₁-Gαs allele and the GPA˜I-SGS allele, but if thisinteraction is somewhat degenerate, then it may be difficult toquantitate this accurately. The second conclusion that can be drawn fromthese results is that there are other determinants involved instabilizing the interaction of Gα with Gβγ beyond these two regions asnone of the GPA1/Gαs hybrid proteins couple as efficiently to yeast Gβγas does native GPA1.

[0211] The role of the surface-exposed residues of this region may becrucial for effective coupling to yeast Gβγ, and can be incorporatedinto hybrid molecules as follows below.

[0212] GαS-GPA-Switch GαS 1-202/GPA298-350/GαS 253-394

[0213] This hybrid encodes the entire switch region of GPA1 in thecontext of GaS.

[0214] GαS-GPA-α2 GQS 1-226/GPA322-332/GQS 238-394

[0215] This hybrid encodes the a² helix of GPA1 in the context of GαS.

[0216] GPA41-GαS-GPA-α2GPA1-41/GQS43-226/GPA322-332/GQS238-394

[0217] This hybrid encodes the 41 residue amino terminal domain of GPA1and the α2 helix of GPA1 in the context of GαS.

[0218] Finally, the last class of hybrids that will be discussed hereare those that alter the surface exposed residues of the β2 and β3sheets of αS so that they resemble those of the GPA1 QS helix. Thesealtered α2 helical domains have the following structure. (The positionsof the altered residues correspond to GαS.)

[0219] L203K

[0220] K211E

[0221] D215G

[0222] K216S

[0223] D229S

[0224] These single mutations can be engineered into a GαS backbonesingly and in pairwise combinations. In addition, they can be introducedin the context of both the full length GαS and the GPA₄₁-GαS hybriddescribed previously. All are predicted to improve the coupling of Gαsubunits to yeast Gβγ subunits by virtue of improved electrostatic andhydrophobic contacts between this region and the regions of Gβ definedby Whiteway and coworkers (Whiteway et al (supra) that define site(s)that interact with GPA1).

[0225] In summary, the identification of hybrid Gα subunits that coupleto the yeast pheromone pathway has led to the following generalobservations. First, all GPA_(BAMH1) hybrids associate with yeast Gβγ,therefore at a minimum these hybrids contain the determinants in GPA1necessary for coupling to the pheromone response pathway. Second, theamino terminal 41 residues of GPA1 contain sufficient determinants tofacilitate coupling of Gα hybrids to yeast Gβγ in some, but not all,instances, and that some Gα subunits contain regions outside of thefirst 41 residues that are sufficiently similar to those in GPA1 tofacilitate interaction with GPA1 even in the absence of the aminoterminal 41 residues of GPA1. Third, there are other determinants in thefirst 310 residues of GPA1 that are involved in coupling Gα subunits toyeast Gβγ subunits.

[0226] The various classes of hybrids noted above are not mutuallyexclusive. For example, a GPA1 containing GPA1-₄₁ could also feature theL203K mutation.

[0227] While, for the sake of simplicity, we have described hybrids ofyeast GPA1 and a mammalian Gαs, it will be appreciated that hybrids maybe made of other yeast Gα subunits and/or other mammalian Gα subunits,notably mammalian Gαi subunits. Moreover, while the described hybridsare constructed from two parental proteins, hybrids of three or moreparental proteins are also possible.

[0228] As shown in the Examples, chimeric Gα subunits have beenespecially useful in coupling receptors to Gαi species.

[0229] G. Expression of Gα

[0230] Kang et al. supra reported that several classes of nativemammalian G˜ subunits were able to interact functionally with yeast asubunits when expression of Gα was driven from a constitutively active,strong promoter (PGK) or from a strong inducible promoter (CUP). Theseauthors reported that rat GαS, Gαi2 or Gα expressed at high levelcoupled to yeast βγ. High level expression of mammalian Gα (i.e.non-stoichiometric with respect to yeast βγ) is not desirable for useslike those described in this application. Reconstruction of Gprotein-coupled receptor signal transduction in yeast requires thesignalling component of the heterotrimeric complex (Gβγ) to be presentstoichiometrically with Gα subunits. An excess of Gα subunits (as wasrequired for coupling of mammalian Gαi2 and Gαo to yeast Gβγ in Kang etal.) would dampen the signal in systems where Gβγ subunits transduce thesignal. An excess of Gα subunits raises the background level ofsignaling in the system to unacceptably high levels. Preferably, levelsof Gα and Gβγ subunits are balanced. For example, heterologous Gαsubunits may be expressed from a low copy (CEN ARS) vector containingthe endogenous yeast GPA1 promoter and the GPA1 3′ untranslated region.The minimum criterion, applied to a heterologous Gαsubunit with respectto its ability to couple functionally to the yeast pheromone pathway, isthat it complement a gpal genotype when expressed from the GPA1 promoteron low copy plasmids or from an integrated, single copy gene. In thework described in this application, all heterologous Gα subunits havebeen assayed in two biological systems. In the first assay heterologousGα subunits are tested for an ability to functionally complement thegrowth arrest phenotype of gpal strains. In the second assay thetranscription of a fus1-HIS3 reporter gene is used to measure the extentto which the pheromone response pathway is activated, and hence theextent to which the heterologous Gα subunit sequesters the endogenousyeast Gβγ complex. Mammalian Gαs, Gαi2, Gαi3, Gαq, Gα11, Gα16, Gαo_(a),Gαo_(b), and Gαz from rat, murine or human origins were expressed from alow copy, CEN ARS vector containing the GPA1 promoter. Functionalcomplementation of gpal strains was not observed in either assay systemwith any of these full-length Gα constructs with the exception of ratand human GαS.

[0231] H. Chimeric Yeast βγ Subunits

[0232] An alternative to the modification of a mammalian Gα subunit forimproved signal transduction is the modification of the pertinent sitesin the yeast Gβ or Gγ subunits. The principles discussed already withrespect to Gα subunits apply, mutatis mutandis, to yeast Gβ or Gγ.

[0233] For example, it would not be unreasonable to target the yeastSte4p Gβsubunit with cassette mutagenesis. Specifically, the region ofSte4p that encodes several of the dominant negative, signaling-defectivemutations would be an excellent target for cassette mutagenesis whenlooking for coupling of yeast Gβγ to specific mammalian Gα subunits.

[0234] X. Peptide Libraries

[0235] While others have engineered yeast cells to facilitate screeningof exogenous drugs as receptor agonists and antagonists, the cells didnot themselves produce both the drugs and the receptors. Yeast cellsengineered to produce the receptor, but that do not produce the drugsthemselves, are inefficient. To utilize them one must bring a sufficientconcentration of each drug into contact with a number of cells in orderto detect whether or not the drug has an action. Therefore, a microtiterplate well or test tube must be used for each drug. The drug must besynthesized in advance and be sufficiently pure to judge its action onthe yeast cells. When the yeast cell-produces the drug, the effectiveconcentration is higher.

[0236] Peptide libraries are systems which simultaneously display, in aform which permits interaction with a target, a highly diverse andnumerous collection of peptides. These peptides may be presented insolution (Houghten (1992) Biotechniques 13:412-421), or on beads (Lam(1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. patent'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869)or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Many of these systems are limited in terms of the maximumlength of the peptide or the composition of the peptide (e.g., Cysexcluded). Steric factors, such as the proximity of a support, mayinterfere with binding. Usually, the screening is for binding in vitroto an artificially presented target, not for activation or inhibition ofa cellular signal transduction pathway in a living cell. While a cellsurface receptor may be used as a target, the screening will not revealwhether the binding of the peptide caused an allosteric change in theconformation of the receptor.

[0237] The Ladner et al. patent, U.S. Pat. No. 5,096,815, describes amethod of identifying novel proteins or polypeptides with a desired DNAbinding activity. Semi-random (“variegated”) DNA encoding a large numberof different potential binding proteins is introduced, in expressibleform, into suitable host cells. The target DNA sequence is incorporatedinto a genetically engineered operon such that the binding of theprotein or polypeptide will prevent expression of a gene product that isdeleterious to the gene under selective conditions. Cells which survivethe selective conditions are thus cells which express a protein whichbinds the target DNA. While it is taught that yeast cells may be usedfor testing, bacterial cells are preferred. The interactions between theprotein and the target DNA occur only in the cell (and then only in thenucleus), not in the periplasm or cytoplasm, and the target is a nucleicacid, and not a receptor protein. Substitution of random peptidesequences for functional domains in cellular proteins permits somedetermination of the specific sequence requirements for theaccomplishment of function. Though the details of the recognitionphenomena which operate in the localization of proteins within cellsremain largely unknown, the constraints on sequence variation ofmitochondrial targeting sequences and protein secretion signal sequenceshave been elucidated using random peptides (Lemire et al., J. Biol.Chem.(1989) 264, 20206 and Kaiser et al. (1987) Science 235:312,respectively).

[0238] The peptide library of the present invention takes the form of acell culture, in which essentially each cell expresses one, and usuallyonly one, peptide of the library. While the diversity of the library ismaximized if each cell produces a peptide of a different sequence, it isusually prudent to construct the library so there is some redundancy.Depending on size, the combinatorial peptides of the library can beexpressed as is, or can be incorporated into larger fusion proteins. Thefusion protein can provide, for example, stability against degradationor denaturation, as well as a secretion signal if secreted. In anexemplary embodiment of a library for intracellular expression, e.g.,for use in conjunction with intracellular target receptors, thepolypeptide library is expressed as thioredoxin fusion proteins (see,for example, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publicationWO94/02502). The combinatorial peptide can be attached one the terminusof the thioredoxin protein, or, for short peptide libraries, insertedinto the so-called active loop.

[0239] In one embodiment, the peptide library is derived to express acombinatorial library of polypeptides which are not based on any knownsequence, nor derived from cDNA. That is, the sequences of the libraryare largely random. In preferred embodiments, the combinatorialpolypeptides are in the range of 3-100 amino acids in length, morepreferably at least 5-50, and even more preferably at least 10, 13, 15,20 or 25 amino acid residues in length. Preferably, the polypeptides ofthe library are of uniform length. It will be understood that the lengthof the combinatorial peptide does not reflect any extraneous sequenceswhich may be present in order to facilitate expression, e.g., such assignal sequences or invariant portions of a fusion protein.

[0240] In another embodiment, the peptide library is derived to expressa combinatorial library of polypeptides which are based at least in parton a known polypeptide sequence or a portion thereof (not a cDNAlibrary). That is, the sequences of the library is semi-random, beingderived by combinatorial mutagenesis of a known sequence. See, forexample, Ladner et al. PCT publication WO 90/02909; Garrard et al., PCTpublication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461. Accordingly, polypeptide(s) which are known ligands for atarget receptor can be mutagenized by standard techniques to derive avariegated library of polypeptide sequences which can further bescreened for agonists and/or antagonists. For example, the surrogateligand identified for FPRL-1, e.g., theSer-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Met peptide, can bemutagenized to generate a library of peptides with some relationship tothe original tridecapeptide. This library can be expressed in a reagentcell of the present invention, and other receptor activators can beisolated from the library. This may permit the identification of evenmore potent FPRL-1 surrogate ligands.

[0241] Alternatively, the library can be expressed under conditionswherein the cells are in contact with the original tridecapeptide, e.g.,the FPRL-1 receptor is being induced by that surrogate ligand. Peptidesfrom an expressed library can be isolated based on their ability topotentiate the induction, or to inhibit the induction, caused by thesurrogate ligand. The latter of course will identify potentialantagonists of chemoattractant receptors. In still other embodiments,the surrogate ligand can be used to screen exogenous compound libraries(peptide and non-peptide) which, by modulating the activity of theidentified surrogate, will presumably also similarly effect the nativeligand's effect on the target receptor. In such embodiments, thesurrogate ligand can be applied to the cells, though is preferablyproduced by the reagent cell, thereby providing an autocrine cell.

[0242] In still another embodiment, the combinatorial polypeptides areproduced from a cDNA library.

[0243] In a preferred embodiment of the present invention, the yeastcells collectively produce a “peptide library”, preferably including atleast 10³ to 10⁷ different peptides, so that diverse peptides may besimultaneously assayed for the ability to interact with the exogenousreceptor. In an especially preferred embodiment, at least some peptidesof the peptide library are secreted into the periplasm, where they mayinteract with the “extracellular” binding site(s) of an exogenousreceptor. They thus mimic more closely the clinical interaction of drugswith cellular receptors. This embodiment optionally may be furtherimproved (in assays not requiring pheromone secretion) by preventingpheromone secretion, and thereby avoiding competition between thepeptide and the pheromone for signal peptidase and other components ofthe secretion system.

[0244] In the present invention, the peptides of the library are encodedby a mixture of DNA molecules of different sequence. Eachpeptide-encoding DNA molecule is ligated with a vector DNA molecule andthe resulting recombinant DNA molecule is introduced into a host cell.Since it is a matter of chance which peptide encoding DNA molecule isintroduced into a particular cell, it is not predictable which peptidethat cell will produce. However, based on a knowledge of the manner inwhich the mixture was prepared, one may make certain statisticalpredictions about the mixture of peptides in the peptide library.

[0245] It is convenient to speak of the peptides of the library as beingcomposed of constant and variable residues. If the nth residue is thesame for all peptides of the library, it is said to be constant. If thenth residue varies, depending on the peptide in question, the residue isa variable one. The peptides of the library will have at least one, andusually more than one, variable residue. A variable residue may varyamong any of two to all twenty of the genetically encoded amino acids;the variable residues of the peptide may vary in the same or differentmanner. Moreover, the frequency of occurrence of the allowed amino acidsat a particular residue position may be the same or different. Thepeptide may also have one or more constant residues.

[0246] There are two principal ways in which to prepare the required DNAmixture. In one method, the DNAs are synthesized a base at a time. Whenvariation is desired, at a base position dictated by the Genetic Code, asuitable mixture of nucleotides is reacted with the nascent DNA, ratherthan the pure nucleotide reagent of conventional polynucleotidesynthesis.

[0247] The second method provides more exact control over the amino acidvariation. First, trinucleotide reagents are prepared, eachtrinucleotide being a codon of one (and only one) of the amino acids tobe featured in the peptide library. When a particular variable residueis to be synthesized, a mixture is made of the appropriatetrinucleotides and reacted with the nascent DNA. Once the necessary“degenerate” DNA is complete, it must be joined with the DNA sequencesnecessary to assure the expression of the peptide, as discussed in moredetail below, and the complete DNA construct must be introduced into theyeast cell.

[0248] XI. Screening and Selection: Assays of Second MessengerGeneration

[0249] When screening for bioactivity of peptides, intracellular secondmessenger generation can be measured directly. A variety ofintracellular effectors have been identified as beingG-protein-regulated, including adenylyl cyclase, cyclic GMP,phosphodiesterases, phosphoinositidase C, and phospholipase A₂. Inaddition, G proteins interact with a range of ion channels and are ableto inhibit certain voltage-sensitive Ca⁺⁺ transients, as well asstimulating cardiac K⁺ channels.

[0250] In one embodiment, the GTPase enzymatic activity by G proteinscan be measured in plasma membrane preparations by determining thebreakdown of γ³²P GTP using techniques that are known in the art (Forexample, see Signal Transduction: A Practical Approach. G. Milligan, Ed.Oxford University Press, Oxford England). When receptors that modulatecAMP are tested, it will be possible to use standard techniques for cAMPdetection, such as competitive assays which quantitate [³H]cAMP in thepresence of unlabelled cAMP.

[0251] Certain receptors stimulate the activity of phospholipase C whichstimulates the breakdown of phosphatidylinositol 4,5, bisphosphate to1,4,5-IP3 (which mobilizes intracellular Ca++) and diacylglycerol (DAG)(which activates protein kinase C). Inositol lipids can be extracted andanalyzed using standard lipid extraction techniques. DAG can also bemeasured using thin-layer chromatography. Water soluble derivatives ofall three inositol lipids (IP1, IP2, IP3) can also be quantitated usingradiolabelling techniques or HPLC.

[0252] The mobilization of intracellular calcium or the influx ofcalcium from outside the cell can be measured using standard techniques.The choice of the appropriate calcium indicator, fluorescent,bioluminescent, metallochromic, or Ca++-sensitive microelectrodesdepends on the cell type and the magnitude and time constant of theevent under study (Borle (1990) Environ Health Perspect 84:45-56). As anexemplary method of Ca++ detection, cells could be loaded with the Ca++sensitive fluorescent dye fura-2 or indo-1, using standard methods, andany change in Ca++ measured using a fluorometer.

[0253] The other product of PIP2 breakdown, DAG can also be producedfrom phosphatidyl choline. The breakdown of this phospholipid inresponse to receptor-mediated signaling can also be measured using avariety of radiolabelling techniques.

[0254] The activation of phospholipase A2 can easily be quantitatedusing known techniques, including, for example, the generation ofarachadonate in the cell.

[0255] In the case of certain receptors, it may be desirable to screenfor changes in cellular phosphorylation. Such assay formats may beuseful when the receptor of interest is a receptor tyrosine kinase. Forexample, yeast transformed with the FGF receptor and a ligand whichbinds the FGF receptor could be screened using colony immunoblotting(Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) usinganti-phosphotyrosine. In addition, tests for phosphorylation could beuseful when a receptor which may not itself be a tyrosine kinase,activates protein kinases that function downstream in the signaltransduction pathway. Likewise, it is noted that protein phosphorylationalso plays a critical role in cascades that serve to amplify signalsgenerated at the receptor. Multi-kinase cascades allow not only signalamplification but also signal divergence to multiple effectors that areoften cell-type specific, allowing a growth factor to stimulate mitosisof one cell and differentiation of another.

[0256] One such cascade is the MAP kinase pathway that appears tomediate both mitogenic, differentiation and stress responses indifferent cell types. Stimulation of growth factor receptors results inRas activation followed by the sequential activation of c-Raf, MEK, andp44 and p42 MAP kinases (ERK1 and ERK2). Activated MAP kinase thenphosphorylates many key regulatory proteins, including p90RSK and Elk-1that are phosphorylated when MAP kinase translocates to the nucleus.Homologous pathways exist in mammalian and yeast cells. For instance, anessential part of the S. cerevisiae pheromone signaling pathway iscomprised of a protein kinase cascade composed of the products of theSTE11, STE7, and FUS3/KSS1 senes (the latter pair are distinct andfunctionally redundant). Accordingly, phosphorylation and/or activationof members of this kinase cascade can be detected and used to quantitatereceptor engagement. Phosphotyrosine specific antibodies are availableto measure increases in tyrosine phosphorylation and phospho-specificantibodies are commercially available (New England Biolabs, Beverly,Mass.).

[0257] Modified methods for detecting receptor-mediated signaltransduction exist and one of skill in the art will recognize suitablemethods that may be used to substitute for the example methods listed.

[0258] XII. Screening and Selection Using Reporter Gene Constructs

[0259] In addition to measuring second messenger production, reportergene constructs can be used. Reporter gene constructs are prepared byoperatively linking a reporter gene with at least one transcriptionalregulatory element. If only one transcriptional regulatory element isincluded it must be a regulatable promoter, At least one the selectedtranscriptional regulatory elements must be indirectly or directlyregulated by the activity of the selected cell-surface receptor wherebyactivity of the receptor can be monitored via transcription of thereporter genes.

[0260] The construct may contain additional transcriptional regulatoryelements, such as a FIRE sequence, or other sequence, that is notnecessarily regulated by the cell surface protein, but is selected forits ability to reduce background level transcription or to amplify thetransduced signal and to thereby increase the sensitivity andreliability of the assay.

[0261] Many reporter genes and transcriptional regulatory elements areknown to those of skill in the art and others may be identified orsynthesized by methods known to those of skill in the art. Reportergenes.

[0262] A reporter gene includes any gene that expresses a detectablegene product, which may be RNA or protein. Preferred reporter genes arethose that are readily detectable. The reporter gene may also beincluded in the construct in the form of a fusion gene with a gene thatincludes desired transcriptional regulatory sequences or exhibits otherdesirable properties.

[0263] Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238,Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secretedalkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol.216:362-368).

[0264] Transcriptional control elements include, but are not limited to,promoters, enhancers, and repressor and activator binding sites.Suitable transcriptional regulatory elements may be derived from thetranscriptional regulatory regions of genes whose expression is rapidlyinduced, generally within minutes, of contact between the cell surfaceprotein and the effector protein that modulates the activity of the cellsurface protein. Examples of such genes include, but are not limited to,the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485),such as c-fos, Immediate early genes are genes that are rapidly inducedupon binding of a ligand to a cell surface protein. The transcriptionalcontrol elements that are preferred for use in the gene constructsinclude transcriptional control elements from immediate early genes,elements derived from other genes that exhibit some or all of thecharacteristics of the immediate early genes, or synthetic elements thatare constructed such that genes in operative linkage therewith exhibitsuch characteristics. The characteristics of preferred genes from whichthe transcriptional control elements are derived include, but are notlimited to, low or undetectable expression in quiescent cells, rapidinduction at the transcriptional level within minutes of extracellularsimulation, induction that is transient and independent of new proteinsynthesis, subsequent shut-off of transcription requires new proteinsynthesis, and mRNAs transcribed from these genes have a shorthalf-life. It is not necessary for all of these properties to bepresent.

[0265] In the most preferred constructs, the transcriptional regulatoryelements are derived from the c-fos gene.

[0266] The c-fos proto oncogene is the cellular homolog of thetransforming gene of FBJ osteosarcoma virus. It encodes a nuclearprotein that most likely involved in normal cellular growth anddifferentiation. Transcription of c-fos is transiently and rapidlyactivated by growth factors and by other inducers of other cell surfaceproteins, including hormones, differentiation-specific agents, stress,mitogens and other known inducers of cell surface proteins. Activationis protein synthesis independent. The c-fos regulatory elements include(see, Verma et al. (1987) Cell 51: a TATA box that is required fortranscription initiation; two upstream elements for basal transcription,and an enhancer, which includes an element with dyad symmetry and whichis required for induction by TPA, serum, EGF, and PMA.

[0267] The 20 bp transcriptional enhancer element located between −317and −298 bp upstream from the c-fos mRNA cap site, which is essentialfor serum induction in serum starved NIH 3T3 cells. One of the twoupstream elements is located at −63-−57 and it resembles the consensussequence for cAMP regulation.

[0268] Other promoters and transcriptional control elements, in additionto those described above, include the vasoactive intestinal peptide(VIP) gene promoter (cAMP responsive; Fink et al. (1988), Proc. Natl.Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMPresponsive; Montminy et al. (1986), Proc. Natl. Acad. Sci.8.3:6682-6686); the proenkephalin promoter (responsive to cAMP,nicotinic agonists, and phorbol esters; Comb et al. (1986), Nature323:353-356); the phosphoenolpyruvate carboxy-kinase gene promoter (cAMPresponsive; Short et al. (1986), J. Biol. Chem. 261:9721-9726); theNGFI-A gene promoter (responsive to NGF, cAMP, and serum; Changelian etal. (1989). Proc. Natl. Acad. Sci. 86:377-381); and others that may beknown to or prepared by those of skill in the art.

[0269] In certain assays it may be desirable to use changes in growth inthe screening procedure. For example, one of the consequences ofactivation of the pheromone signal pathway in wild-type yeast is growtharrest. If one is testing for an antagonist of a G protein-coupledreceptor, this normal response of growth arrest can be used to selectcells in which the pheromone response pathway is inhibited. That is,cells exposed to both a known agonist and a peptide of unknown activitywill be growth arrested if the peptide is neutral or an agonist, butwill grow normally if the peptide is an antagonist. Thus, the growtharrest response can be used to advantage to discover peptides thatfunction as antagonists.

[0270] However, when searching for peptides which can function asagonists of G protein-coupled receptors, or other pheromone systemproteins, the growth arrest consequent to activation of the pheromoneresponse pathway is an undesirable effect since cells that bind peptideagonists stop growing while surrounding cells that fail to bind peptideswill continue to grow. The cells of interest, then, will be overgrown ortheir detection obscured by the background cells, confoundingidentification of the cells of interest. To overcome this problem thepresent invention teaches engineering the cell such that: 1) growtharrest does not occur as a result of exogenous signal pathway activation(e.g., by inactivating the FAR1 gene); and/or 2) a selective growthadvantage is conferred by activating the pathway (e.g., by transformingan auxotrophic mutant with a HIS3 gene under the control of apheromone-responsive promoter, and applying selective conditions).

[0271] It is, of course, desirable that the exogenous receptor beexposed on a continuing basis to the peptides. Unfortunately, this islikely to result in desensitization of the pheromone pathway to thestimulus. For example, the mating signal transduction pahtway is knownto become desensitized by several mechanisms including pheromonedegradation and modification of the function of the receptor, Gproteins,s and/or downstream elements of the pheromone signaltransduction by the products of the SST2, STE50, AFR1 (Konopka, J. B.(1993) Mol. Cell. Biol. 13:6876-6888) and SGV1, MSG5, and SIG1 genes.Selected mutations in these genes can lead to hypersensitivity topheromone and an inability to adapt to the presence of pheromone. Forexample, introduction of mutations that interfere with function intostrains expressing heterologous G protein-coupled receptors constitutesa significant improvement on wild type strains and enables thedevelopment of extremely sensitive bioassays for compounds that interactwith the receptors. Other mutations e.g. STE50, sgv1, bar1, ste2,ste3,pik1, msg5, sig1, and aft1, have the similar effect of increasing thesensitivity of the bioassay. Thus desensitization may be avoided bymutating (which may include deleting) the SST2 gene so that it no longerproduces a functional protein, or by mutating one of the other geneslisted above.

[0272] If the endogenous homolog of the receptor is produced by theyeast cell, the assay will not be able to distinguish between peptideswhich interact with the endogenous receptor and those which interactwith the exogenous receptor. It is therefore desirable that theendogenous gene be deleted or otherwise rendered nonfunctional.

[0273] In the case of receptors which modulate cyclic AMP, atranscriptional based readout can be constructed using the cyclic AMPresponse element binding protein, CREB, which is a transcription factorwhose activity is regulated by phosphorylation at a particular serine(S133). When this serine residue is phosphorylated, CREB binds to arecognition sequence known as a CRE (cAMP Responsive Element) found tothe 5′ of promotors known to be responsive to elevated cAMP levels. Uponbinding of phosphorylated CREB to a CRE, transcription from thispromoter is increased.

[0274] Phosphorylation of CREB is seen in response to both increasedcAMP levels and increased intracellular Ca levels. Increased cAMP levelsresult in activation of PKA, which in turn phosphorylates CREB and leadsto binding to CRE and transcriptional activation. Increasedintracellular calcium levels results in activation of calcium/calmodulinresponsive kinase IV (CaM kinase IV). Phosphorylation of CREB by CaMkinase IV is effectively the same as phosphorylation of CREB by PKA, andresults in transcriptional activation of CRE containing promotors.

[0275] Therefore, a transcriptional-based readout can be constructed incells containing a reporter gene whose expression is driven by a basalpromoter containing one or more CRE. Changes in the intracellularconcentration of Ca⁺⁺ (a result of alterations in the activity of thereceptor upon engagement with a ligand) will result in changes in thelevel of expression of the reporter gene if: a) CREB is alsoco-expressed in the cell, and b) either the endogenous yeast CaM kinasewill phosphorylate CREB in response to increases in calcium or if anexogenously expressed CaM kinase IV is present in the same cell. Inother words, stimulation of PLC activity will result in phosphorylationof CREB and increased transcription from the CRE-construct, whileinhibition of PLC activity will result in decreased transcription fromthe CRE-responsive construct.

[0276] As described in Bonni et al. (1993) Science 262:1575-1579, theobservation that CNTF treatment of SK-N-MC cells leads to the enhancedinteraction of STAT/p91 and STAT related proteins with specific DNAsequences suggested that these proteins might be key regulators ofchanges in gene expression that are triggered by CNTF. Consistent withthis possibility is the finding that DNA sequence elements similar tothe consensus DNA sequence required for STAT/p91 binding are presentupstream of a number of genes previously found to be induced by CNTF(e.g., Human c-fos, Mouse c-fos, Mouse tis11, Rat junB, Rat SOD-1, andCNTF). Those authors demonstrated the ability of STAT/p91 binding sitesto confer CNTF responsiveness to a non-responsive reporter gene.Accordingly, a reporter construct for use in the present invention fordetecting signal transduction through STAT proteins, such as fromcytokine receptors, can be generated by using −71 to +109 of the mousec-fos gene fused to the bacterial chloramphenicol acetyltransferase gene(−71fosCAT) or other detectable marker gene. Induction by a cytokinereceptor induces the tyrosine phosphorylation of STAT and STAT-relatedproteins, with subsequent translocation and binding of these proteins tothe STAT-RE. This then leads to activation of transcription of genescontaining this DNA element within their promoters.

[0277] In preferred embodiments, the reporter gene is a gene whoseexpression causes a phenotypic change which is screenable or selectable.If the change is selectable, the phenotypic change creates a differencein the growth or survival rate between cells which express the reportergene and those which do not. If the change is screenable, the phenotypechange creates a difference in some detectable characteristic of thecells, by which the cells which express the marker may be distinguishedfrom those which do not. Selection is preferable to screening in that itcan provide a means for amplifying from the cell culture those cellswhich express a test polypeptide which is a receptor effector.

[0278] The marker gene is coupled to the receptor signaling pathway sothat expression of the marker gene is dependent on activation of thereceptor. This coupling may be achieved by operably linking the markergene to a receptor-responsive promoter. The term “receptor-responsivepromoter” indicates a promoter which is regulated by some product of thetarget receptor's signal transduction pathway.

[0279] Alternatively, the promoter may be one which is repressed by thereceptor pathway, thereby preventing expression of a product which isdeleterious to the cell. With a receptor repressed promoter, one screensfor agonists by linking the promoter to a deleterious gene, and forantagonists, by linking it to a beneficial gene. Repression may beachieved by operably linking a receptor-induced promoter to a geneencoding mRNA which is antisense to at least a portion of the mRNAencoded by the marker gene (whether in the coding or flanking regions),so as to inhibit translation of that mRNA. Repression may also beobtained by linking a receptor-induced promoter to a gene encoding a DNAbinding repressor protein, and incorporating a suitable operator siteinto the promoter or other suitable region of the marker gene.

[0280] In the case of yeast, suitable positively selectable (beneficial)genes include the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1, 2, 3,4, 5, 7, 8; ARG1, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2, 5; THR1, 4; TRP2,3, 4, 5; LEU1, 4; MET2, 3, 4, 8, 9, 14, 16, 19; URA1, 2, 4, 5, 10; HOM3,6; ASP3; CHO1; ARO2, 7; CYS3; OLE1; INO1, 2, 4; PRO1, 3 Countless othergenes are potential selective markers. The above are involved inwell-characterized biosynthetic pathways. The imidazoleglycerolphosphate dehydratase (IGP dehydratase) gene (HIS3) is preferred becauseit is both quite sensitive and can be selected over a broad range ofexpression levels. In the simplest case, the cell is auxotrophic forhistidine (requires histidine for growth) in the absence of activation.Activation leads to synthesis of the enzyme and the cell becomesprototrophic for histidine (does not require histidine). Thus theselection is for growth in the absence of histidine. Since only a fewmolecules per cell of IGP dehydratase are required for histidineprototrophy, the assay is very sensitive.

[0281] In a more complex version of the assay, cells can be selected forresistance to aminotriazole (AT), a drug that inhibits the activity ofIGP dehydratase. Cells with low, fixed level of expression of HIS3 aresensitive to the drug, while cells with higher levels are resistant. Theamount of AT can be selected to inhibit cells with a basal level of HIS3expression (whatever that level is) but allow growth of cells with aninduced level of expression. In this case selection is for growth in theabsence of histidine and in the presence of a suitable level of AT.

[0282] In appropriate assays, so-called counterselectable or negativelyselectable genes may be used. Suitable genes include: URA3(orotidine-5′-phosphate decarboxylase; inhibits growth on 5-fluorooroticacid), LYS2 (2-aminoadipate reductase; inhibits growth on α-aminoadipateas sole nitrogen source), CYH2 (encodes ribosomal protein L29;cycloheximide-sensitive allele is dominant to resistant allele), CAN1(encodes arginine permease; null allele confers resistance to thearginine analog canavanin), and other recessive drug-resistant markers.

[0283] In one example, the marker gene effects yeast cell growth. Thenatural response to signal transduction via the yeast pheromone systemresponse pathway is for cells to undergo growth arrest. This is thepreferred way to select for antagonists to a ligand/receptor pair thatinduces the pathway. An autocrine peptide antagonist would inhibit theactivation of the pathway; hence, the cell would be able to grow. Thus,the FAR1 gene may be considered an endogenous counterselectable marker.The FAR1 gene is preferably inactivated when screening for agonistactivity.

[0284] The marker gene may also be a screenable gene. The screenedcharacteristic may be a change in cell morphology, metabolism or otherscreenable features. Suitable markers include beta-galactosidase (Xga1,C₁₂FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkalinephosphatase, horseradish peroxidase, exo-glucanase (product of yeastexb1 gene; nonessential, secreted); luciferase; bacterial greenfluorescent protein; (human placental) secreted alkaline phosphatase(SEAP); and chloramphenicol transferase (CAT). Some of the above can beengineered so that they are secreted (although not β-galactosidase). Apreferred screenable marker gene is beta-galactosidase; yeast cellsexpressing the enzyme convert the colorless substrate Xga1 into a bluepigment. Again, the promoter may be receptor-induced orreceptor-inhibited.

[0285] XIII. Genetic Markers in Yeast Strains

[0286] Yeast strains that are auxotrophic for histidine (HIS3) areknown, see Struhl and Hill, (1987) Mol. Cell. Biol., 7:104; Fasullo andDavis, Mol. Cell. Biol., (1988) 8:4370. The HIS3 (imidazoleglycerolphosphate dehydratase) gene has been used as a selective marker inyeast. See Sikorski and Heiter, (1989) Genetics, 122:19; Struhl, et al.,P.N.A.S. (1979) 76:1035; and, for FUS1-HIS3 fusions, see Stevenson, etal., (1992) Genes Dev., 6:1293.

[0287] XIV Novel FPRL-1 Ligand

[0288] Yet another aspect of the invention pertains to a novel ligandfor the orphan receptor, FPRL-1. As described in Example 8, atridecapeptide having the sequenceSer-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Met was identified froma polypeptide library on the basis of its ability to act as a surrogateligand for FPRL-1.

[0289] Chemoattractants are important mediators of inflammation, theyfunction to recruit phagocytic cells at the site of injury or infection.They also mediate granule secretion, superoxide generation andupregulation of cell surface adhestion molecules, for example MAC-1.Exemplary chemoattractants include the complement component C5a,interleukin 8, leukotriene B4 and platelet activiating factor. Many ofthese substances participate in pathophysiological conditions such asanaphylaxis and septic shock. The identification of ligands for theorphan FPRL1 receptor provides new opportunities for discovery ofreceptor agonists, that could potentially serve to enhance lymphocyterecruitment in immunocompromised patients, and for the discovery ofreceptor antagonists (described supra) that could prevent undesirableconsequences of immune activation such as anaphylactic or septic shock.

[0290] The term “peptide” is used herein to refer to a chain of two ormore amino acids or amino acid analogs (including non-naturallyoccurring amino acids), with adjacent amino acids joined by peptide(—NHCO—) bonds. Thus, the peptides of the present invention includeoligopeptides, polypeptides, and proteins. Preferably, the peptides ofthe present invention include all or a portion of theS-L-L-W-L-T-C-R-P-W-E-A-M peptide, or a bomolog thereof. The peptide (orpeptidomimetic) is preferably at least 3 amino acid residues in length,though peptides of up to 13 amino acids, such as 4, 5, 7, 10, 13 or moreresidues in length, are preferred. Longer peptides which include theFPRL ligand are also contemplated. For example, the sequence derivedfrom the FPRL-1 surrogate ligand can be provided as part of a fusionprotein. The minimum peptide length is chiefly dictated by the need toobtain sufficient potency as an activator or inhibitor. Given the sizeof the peptide isolated in subject assay, smaller fragments of thetridecapeptide which retain receptor binding activity will be easilyidentified, e.g., by chemical synthesis of different fragments. Themaximum peptide length will only be a function of synthetic convenienceonce an active peptide is identified.

[0291] The invention also provides for the generation of mimetics, e.g.peptide or non-peptide agents. Moreover, the present invention alsocontemplates variants of the subject polypeptide which may themselves beeither agonistic or antagonistic of the S-L-L-W-L-T-C-R-P-W-E-A-Mpeptide. Thus, using such mutagenic techniques as known in the art, thedeterminants of S-L-L-W-L-T-C-R-P-W-E-A-M polypeptide which participatein FPRL-1 interactions can be ellucidated. To illustrate, the criticalresidues of a subject polypeptide which are involved in molecularrecognition of an FPRL-1 receptor can be determined and used to generatevariant polypeptides which competitively inhibit binding of theauthentic S-L-L-W-L-T-C-R-P-W-E-A-M peptide with that receptor. Byemploying, for example, scanning mutagenesis to map the amino acidresidues of the polypeptide involved in binding the FPRL-1 receptor,peptide and peptidomimetic compounds can be generated which mimic thoseresidues in binding to the receptor and which consequently can inhibitbinding of an authentic ligand for the FPRL-1 receptor and interferewith the function of that receptor.

[0292] Moreover, as is apparent from the present and parent disclosures,mimetopes of the subject S-L-L-W-L-T-C-R-P-W-E-A-M peptide can beprovided as non-hydrolyzable peptide analogs. For illustrative purposes,peptide analogs of the present invention can be generated using, forexample, benzodiazepines (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p123), C-7 mimics (Huffman et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), β-aminoalcohols (Gordonet al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al.(1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modifed(Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p134). Also, see generally,Session III: Analytic and synthetic methods, in in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988)

[0293] In an exemplary embodiment, the peptidomimetic can be derived asa retro-inverso analog of the peptide. To illustrate, theS-L-L-W-L-T-C-R-P-W-E-A-M peptide can be generated as the retro-inversoanalog:

[0294] Such retro-inverso analogs can be made according to the methodsknown in the art, such as that described by the Sisto et al. U.S. Pat.No. 4,522,752. For example, the illustrated retro-inverso analog can begenerated as follows. The geminal diamine corresponding to the serineanalog is synthesized by treating a protected serine with ammonia underHOBT-DCC coupling conditions to yield the N-Boc amide, and theneffecting a Hofmann-type rearrangement withI,I-bis-(trifluoroacetoxy)iodobenzene (TIB), as described inRadhakrishna et al. (1979) J. Org. Chem. 44:1746. The product amine saltis then coupled to a side-chain protected (e.g., as the benzyl ester)N-Fmoc D-Leu residue under standard conditions to yield thepseudodipeptide. The Fmoc (fluorenylmethoxycarbonyl) group is removedwith piperidine in dimethylformamide, and the resulting amine istrimethylsilylated with bistrimethylsilylacetamide (BSA) beforecondensation with suitably alkylated, side-chain protected derivative ofMeldrum's acid, as described in U.S. Pat. No. 5,061,811 to Pinori etal., to yield the retro-inverso tripeptide analog S-L-L. Thepseudotripeptide is then coupled with L-Trp under standard conditions togive the protected tetrapeptide analog. The protecting groups areremoved to release the product, and the steps repeated to enlogate thetetrapeptide to the full length peptide. It will be understood that amixed peptide, e.g. including some normal peptide linkages, can begenerated. As a general guide, sites which are most susceptible toproteolysis are typically altered, with less susceptible amide linkagesbeing optional for mimetic switching The final product, or intermediatesthereof, can be purified by HPLC.

[0295] In another illustrative embodiment, the peptidomimetic can bederived as a retro-enatio analog of the peptide, such as the exemplaryretro-enatio peptide analog derived for the illustrativeS-L-L-W-L-T-C-R-P-W-E-A-M peptide:

[0296] Retro-enantio analogs such as this can be synthesized usingcommercially available D-amino acids and standard solid- orsolution-phase peptide-synthesis techniques. For example, in a preferredsolid-phase synthesis method, a suitably amino-protected(t-butyloxycarbonyl, Boc) D-Serine residue (or analog thereof) iscovalently bound to a solid support such as chloromethyl resin. Theresin is washed with dichloromethane (DCM), and the BOC protecting groupremoved by treatment with TFA in DCM. The resin is washed andneutralized, and the next Boc-protected D-amino acid (D-Leu) isintroduced by coupling with diisopropylcarbodiimide. The resin is againwashed, and the cycle repeated for each of the remaining amino acids inturn (D-Leu, D-Trp etc). When synthesis of the protected retro-enantiopeptide is complete, the protecting groups are removed and the peptidecleaved from the solid support by treatment with hydrofluoricacid/anisole/dimethyl sulfide/thioanisole. The final product is purifiedby HPLC to yield the pure retro-enantio analog.

[0297] In still another illustrative embodiment, trans-olefinderivatives can be made for the subject polypeptide. For example, anexemplary olefin analog is derived for the illustrativeS-L-L-W-L-T-C-R-P-W-E-A-M peptide:

[0298] The trans olefin analog of the subject peptide can be synthesizedaccording to the method of Y. K. Shue et al. (1987) Tetrahedron Letters28:3225.

[0299] Still another class of peptidomimetic derivatives include thephosphonate derivatives, such as the partially phosphonate derivativedS-L-L-W-L-T-C-R-P-W-E-A-M peptide:

[0300] The synthesis of such phosphonate derivatives can be adapted fromknown synthesis schemes. See, for example, Loots et al. in Peptides:Chemistry and Biology, (Escom Science Publishers, Leiden, 1988, p. 118);Petrillo et al. in Peptides: Structure and Function (Proceedings of the9th American Peptide Symposium, Pierce Chemical Co. Rockland, Ill.,1985).

[0301] XV. Novel C5a Ligands

[0302] Still another aspect of the invention pertains to a novel ligandfor the C5a receptor. As described in Example 12, several 13-mer and11-mer peptides have been identified from a polypeptide library on thebasis of their ability to act as surrogate ligands for the C5a receptor.The sequence for exemplary C5a receptor ligands is provided in FIG. 7.Yet another preferred C5a ligand includes all or a portion of thepeptide Asp-Thr-Arg-Ser-Trp-Lys-Leu-Arg-Leu-Leu-Trp-Leu-Ala, describedin the appended examples.

[0303] The importance of the C5a receptor finds its origin in itsrelationship with complement derived C5a and its role in the overallimmune response. In man, and in most animals, the complement system iscomposed of a group of proteins that are normally present in serum in aninactive state. When activated, these proteins participate in acoordinated series of reactions. Activation of the complement systemresults in enzymatic cleavage of complement proteins producingsubfragments which possess a wide range of biologic activities requiredfor host defense, including bloodclotting and inflammatory responses, aswell as activation of immune response directed to the elimination ofinvading microorganisms. During an inflammatory process, localproduction of complement-derived mediators result in increased vascularpermeability, leukocyte adherence to endothelial and vascular tissue,and a chemotactic gradient that induces neutrophil (PMN) migration intothe inflammatory site. In addition to beneficial aspects of theinflammatory process, systemic and/or chronic inflammatory processeshave been associated with a variety of immune disease states. Theanaphylatoxin C5a is one of the best described and most potentproinflammatory mediators derived from the complement system. C5a hasbeen shown to be spasmogenic (Stimler et al. (1981) J. Immunol.126:2258), chemotactic (Hugli et al. (1978) Adv. Immunol. 26:1), toincrease vascular permeability (Shin et al. (1968) Science 162:361), andto induce the release of pharmacologically active mediators fromnumerous cell types (Grant et al. (1975) J. Immunol. 114:1101; Goldsteinet al. (1973) J. Immunol. 113:1583; Schorlemmer et al. (1976) Nature261:48). Most recently, C5a has been shown to directly or indirectlyinduce cytokine release from macrophages and to augment humoral- andcell-mediated immune responses in vitro. Combined, these studiesindicate that C5a possesses multiple biologic activities important inhost defense and may also play a role in inflammatory disease processes.Many cell types possess receptors for C5a, including PMNs, macrophages,mast cells and platelets.

[0304] Among the various cell types, the neutrophil response to C5a isthe best defined. Cell surface receptors specific for C5a have beendemonstrated on the neutrophil (Chenoweth et al. (1978) PNAS 75:3943;Huey et al. (1985) J. Immunol. 135:2063; Rollins et al. (1985) J. Biol.Chem. 260:7157), and the ligand-receptor interaction has been shown topromote human polymorphonuclear leukocyte (PMN) migration in a directedfashion (chemotaxis), adherence, oxidative burst, and granular enzymerelease from these cells (Hugli et al. (1984) Springer Semin.Immunopathol. 7:193). The interaction of C5a with PMN and other targetcells and tissues results in increased histamine release, vascularpermeability, smooth muscle contraction, and an influx into tissues ofinflammatory cells, including neutrophils, eosinophils, and basophils(Hugli et al., supra). C5a may also be important in mediatinginflammatory effects of phagocytic mononuclear cells that accumulate atsites of chronic inflammation (Allison et al. (1978) Agents and Actions8:27). C5a and C5a des-Arg can induce chemotaxis in monocytes (Ward etal. (1968) J. Exp. Med 128:1201. Snyderman et al. (1979) J. Immunol.109:896) and cause them to release lysosomal enzymes in a manneranalogous to the neutrophil responses elicited by these agents. Otherstudies suggest that C5a may have an immunoregulatory role by enhancingantibody particularly at sites of inflammation (Morgan et al. (1982) J.Exp. Med. 155:1412; Weigle et al. (1982) Federation Proc. 41:3099; andMorgan et al. (1984) Federation Proc. 43:2543).

[0305] Accordingly, the peptides identified by the instant assay as C5aligands can be used therapeutically to enhance inflammatory responses.As above, the term “peptide” is used herein to refer to a chain of twoor more amino acids or amino acid analogs (including non-naturallyoccurring amino acids), with adjacent amino acids joined by peptide(—NHCO—) bonds. Thus, the peptides of the present invention includeoligopeptides, polypeptides, and proteins. The peptide (orpeptidomimetic) is preferably at least 3 amino acid residues in length,though peptides of any length up to 13, including peptides of 4, 5, 7,10, 13 or more residues in length are preferred. Longer peptides arealso specifically contemplated. For example, the sequence derived fromthe C5a surrogate ligand can be provided as part of a fusion protein.The minimum peptide length is chiefly dictated by the need to obtainsufficient potency and selectivity as an activator or inhibitor. Giventhe size of the peptide isolated in subject assay, smaller fragments ofthe 11-mer and 13-mer peptides which retain C5a receptor bindingactivity will be easily identified, e.g., by chemical synthesis ofdifferent fragments. The maximum peptide length will only be a functionof synthetic convenience once an active peptide is identified.

[0306] The invention also provides for the generation of mimetics, e.g.peptide or non-peptide agents, of the subject C5a receptor ligands.Moreover, the present invention also contemplates variants of thesubject C5a ligands which may themselves be either agonistic orantagonistic of the C5a receptor activity. Thus, using such mutagenictechniques as known in the art, the determinants of peptide whichparticipate in interaction with the C5a receptor can be ellucidated. Toillustrate, the critical residues of a subject polypeptide which areinvolved in molecular recognition of a C5a receptor can be determinedand used to generate variant polypeptides which competitively inhibitbinding of the original peptide with that receptor. By employing, forexample, scanning mutagenesis to map the amino acid residues of thepolypeptide involved in binding the C5a receptor, peptide andpeptidomimetic compounds can be generated which mimic those residues inbinding to the receptor and which consequently can inhibit binding of anauthentic ligand for the C5a receptor and interfere with the function ofthat receptor. Such C5a receptor antagonists can be useful as inhibitorsof inflammation, e.g., in the treatment of anaphylaxis.

[0307] Moreover, as is apparent from the present and parent disclosures,mimetopes of the subject C5a ligands can be provided as non-hydrolyzablepeptide analogs. For illustrative purposes, peptide analogs of thepresent invention can be generated using, for example, benzodiazepines(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p123), C-7mimics (Huffman et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231),β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71),diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun124:141), and methyleneamino-modifed (Roark et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988, p134). Also, see generally, Session III: Analytic andsynthetic methods, in in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988).

[0308] XVI. Further Manipulation of Peptide Ligands

[0309] The above examples provide guidance for a variety of techniquesfor manipulating peptide ligands indentified in the present screeningassay in order to develop more specific and/or potent agonists orantagonists. In addition, a variety of combinatorial techniques areknown in the art and will be useful for further optimization of thepeptide leads coming of the instant assay. For example, alanine scanningmutagenesis and the like (Lowman et al. (1991) Biochemistry30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085), bylinker scanning mutagenesis (Brown et al. (1992) Mol. Cell Biol.12:2644-2652; McKnight et al. (1982) Science 232:316); by saturationmutagenesis (Meyers et al. (1986) Science 232:613); by PCR mutagenesis(Leung et al. (1989) Method Cell Mol Biol 1:11-19); or by randommutagenesis (Miller et al. (1992) A Short Course in Bacterial Genetics,CSHL Press, Cold Spring Harbor, N.Y.) can be used to create libraries ofvariants which can be further screened, even by simple receptor bindingassays, for receptor binding activity. To further illustrate the stateof the art, it is noted that the review article of Gallop et al. (1994)J Med Chem 37:1233 describe the general state of the art ofcombinatorial libraries. In particular, Gallop et al state at page 1239“[s]creening the analog libraries aids in determining the minimum sizeof the active sequence and in identifying those residues critical forbinding and intolerant of substitution”.

[0310] For the most part, the amino acids used in the subject receptoragonists and antagonists of this invention will be those naturallyoccurring amino acids found in proteins, or the naturally occurringanabolic or catabolic products of such amino acids which contain aminoand carboxyl groups. Particularly suitable amino acid side chainsinclude side chains selected from those of the following amino acids:glycine, alanine, valine, cysteine, leucine, isoleucine, serine,threonine, methionine, glutamic acid, aspartic acid, glutamine,asparagine, lysine, arginine, proline, histidine, phenylalanine,tyrosine, and tryptophan.

[0311] However, the term amino acid residue further includes analogs,derivatives and congeners of any specific amino acid referred to herein.For example, the present invention contemplates the use of amino acidanalogs wherein a side chain is lengthened or shortened while stillproviding a carboxyl, amino or other reactive precursor functional groupfor cyclization, as well as amino acid analogs having variant sidechains with appropriate functional groups). For instance, the subjectpeptidomimetic can include an amino acid analog as for example,b-cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine,homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan,1-methylhistidine, or 3-methylhistidine. Other naturally occurring aminoacid metabolites or precursors having side chains which are suitableherein will be recognized by those skilled in the art and are includedin the scope of the present invention.

[0312] Also included are the D and L stereoisomers of such amino acidswhen the structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols D, L or DL, furthermore when theconfiguration is not designated the amino acid or residue can have theconfiguration D, L or DL. It will be noted that the structure of some ofthe compounds of this invention includes asymmetric carbon atoms. It isto be understood accordingly that the isomers arising from suchasymmetry are included within the scope of this invention. Such isomersare obtained in substantially pure form by classical separationtechniques and by sterically controlled synthesis. For the purposes ofthis application, unless expressly noted to the contrary, a named aminoacid shall be construed to include both the D or L stereoisomers,preferably the L stereoisomer.

[0313] XVII. Pharmaceutical Preparations of Identified Agents

[0314] After identifying certain test compounds as potential surrogateligands, or receptor antagonists, the practioner of the subject assaywill continue to test the efficacy and specificity of the selectedcompounds both in vitro and in vivo. Whether for subsequent in vivotesting, or for administration to an animal as an approved drug, agentsidentified in the subject assay can be formulated in pharmaceuticalpreparations for in vivo administration to an animal, preferably ahuman.

[0315] The compounds selected in the subject assay, or apharmaceutically acceptable salt thereof, may accordingly be formulatedfor administration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thecompound, its use in the pharmaceutical preparation of the invention iscontemplated. Suitable vehicles and their formulation inclusive of otherproteins are described, for example, in the book Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences. MackPublishing Company, Easton, Pa., USA 1985). These vehicles includeinjectable “deposit formulations”. Based on the above, suchpharmaceutical formulations include, although not exclusively, solutionsor freeze-dried powders of the compound in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered media at a suitable pH and isosmotic with physiological fluids.In preferred embodiment, the compound can be disposed in a sterilepreparation for topical and/or systemic administration. In the case offreeze-dried preparations, supporting excipients such as, but notexclusively, mannitol or glycine may be used and appropriate bufferedsolutions of the desired volume will be provided so as to obtainadequate isotonic buffered solutions of the desired pH. Similarsolutions may also be used for the pharmaceutical compositions ofcompounds in isotonic solutions of the desired volume and include, butnot exclusively, the use of buffered saline solutions with phosphate orcitrate at suitable concentrations so as to obtain at all times isotonicpharmaceutical preparations of the desired pH, (for example, neutralpH).

Exemplification

[0316] The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

EXAMPLE 1 Development of Autocrine Yeast Strains

[0317] In this example, we describe a pilot experiment in which haploidcells were engineered to be responsive to their own pheromones. (Notethat in the examples, functional genes are capitalized and inactivatedgenes are in lower case.) For this purpose we constructed recombinantDNA molecules designed to:

[0318] i. place the coding region of STE2 under the transcriptionalcontrol of elements which normally direct the transcription of STE3.This is done in a plasmid that allows the replacement of genomic STE3 ofS. cerevisiae with sequences wherein the coding sequence of STE2 isdriven by STE3 transcriptional control elements.

[0319] ii. place the coding region of STE3 under the transcriptionalcontrol of elements which normally direct the transcription of STE2.This is done in a plasmid which will allow the replacement of genomicSTE2 of S. cerevisiae with sequences wherein the coding sequence of STE3is driven by STE2 transcriptional control elements.

[0320] The sequence of the STE2 gene is known see Burkholder A. C. andHartwell L. H. (1985), Nuc. Acids Res. 13, 8463; Nakayama N., MiyajimaA., Arai K. (1985) EMBO J. 4, 2643.

[0321] A 4.3 kb BamHI fragment that contains the entire STE2 gene wasexcised from plasmid YEp24-STE2 (obtained from J. Thorner, Univ. ofCalifornia) and cloned into pALTER (Protocols and Applications Guide,1991, Promega Corporation, Madison, Wis.). An SpeI site was introduced 7nucleotides (nts) upstream of the ATG of STE2 with the followingmutagenic oligonucleotide, using the STE2 minus strand as template:5′-GTTAAGAACCATATACTAGTATCAAAAATGTCTG 3′

[0322] A second SpeI site was simultaneously introduced just downstreamof the STE2 stop codon with the following mutagenic oligonucleotide:5′-TGATCAAAATTTACTAGTTTGAAAAAGTAATTTCG 3′

[0323] The BamHI fragment of the resulting plasmid (Cadus 1096)containing STE2 with Spel sites immediately flanking the coding region,was then subcloned into the yeast integrating vector YIp19 to yieldCadus 1143.

[0324] The STE3 sequence is also known (Nakayama N., Miyajima A., AraiK. (1985), EMBO J. 4, 2643; (Hagen D. C., McCaffrey G., Sprague G. F.(1986), Proc. Natl. Acad. Sci. 83, 1418. STE3 was made available by Dr.J. Broach as a 3.1 kb fragment cloned into pBLUESCRIPT-KS II(Stratagene, 1011 North Torrey Pines Road, La Jolla, Calif. 92037). STE3was subcloned as a KpnI-XbaI fragment into both M13mp18 RF (to yieldCadus 1105 and pUC19 (to yield Cadus 1107). The two SpeI sites in Cadus1107 were removed by digestion with Spel, fill-in with DNA polymerase IKlenow fragment, and recircularization by blunt-end ligation.Single-stranded DNA containing the minus strand of STE3 was obtainedusing Cadus 1105 and Spel sites were introduced 9 nts upstream of thestart codon and 3 nts downstream of the stop codon of STE3 with thefollowing mutagenic oligonucleotides, respectively:5′-GGCAAAATACTAGTAAAATTTTCATGTC 3′ 5′-GGCCCTTAACACACTAGTGTCGCATTATATTTAC3′

[0325] The mutagenesis was accomplished using the T7-GEN protocol ofUnited States Biochemical (T7-GEN In Vitro Mutagenesis Kit, Descriptionsand Protocols, 1991, United States Biochemical, P.O. Box 22400,Cleveland, Ohio 44122). The replicative form of the resulting Cadus 1141was digested with AflII and KpnI, and the approximately 2 kb fragmentcontaining the entire coding region of STE3 flanked by the two newlyintroduced Spe I sites was isolated and ligated with the approximately3.7 kb vector fragment of AflII- and KpnI-digested Cadus 1107, to yieldCadus 1138. Cadus 1138 was then digested with XbaI and KpnI, and theSTE3-containing 2.8 kb fragment was ligated into the XbaI- andKpnI-digested yeast integrating plasmid pRS406 (Sikorski, R. S. andHieter, P. (1989), Genetics 122:19-27) to yield Cadus 1145.

[0326] The SpeI fragment of Cadus 1143 was replaced with the SpeIfragment of Cadus 1145 to yield Cadus 1147, in which the codingsequences of STE3 are under the control of STE2 expression elements.Similarly, the SpeI fragment of Cadus 1145 was replaced with the Spelfragment of Cadus 1143 to yield Cadus 1148, in which the codingsequences of STE2 are under the control of STE3 expression elements.Using the method of pop-in/pop-out replacement (Rothstein, R. (1991)Methods in Enzymology, 194:281 301), Cadus 1147 was used to replacegenomic STE2 with the ste2-STE3 hybrid in a MATα cell and Cadus 1148 wasused to replace genomic STE3 with the ste3-STE2 hybrid in a MATα cell.Cadus 1147 and 1148 contain the selectable marker URA3.

[0327] Haploid yeast of mating type a which had been engineered toexpress HIS3 under the control of the pheromone-inducible FUS1 promoterwere transformed with CADUS 1147, and transformants expressing URA3 wereselected. These transformants, which express both Ste2p and Ste3p, wereplated on 5-fluoroorotic acid to allow the selection of clones which hadlost the endogenous STE2, leaving in its place the heterologous,integrated STE3. Such cells exhibited the ability to grow on mediadeficient in histidine, indicating autocrine stimulation of thepheromone response pathway.

[0328] Similarly, haploids of mating type α that can express HIS3 underthe control of the pheromone-inducible FUS1 promoter were transformedwith CADUS 1148 and selected for replacement of their endogenous STE3with the integrated STE2. Such cells showed, by their ability to grow onhistidine-deficient media, autocrine stimulation of the pheromoneresponse pathway.

EXAMPLE 2 Strain Development

[0329] In this example, yeast strains are constructed which willfacilitate selection of clones which exhibit autocrine activation of thepheromone response pathway. To construct appropriate yeast strains, wewill use: the YIp-STE3 and pRS-STE2 knockout plasmids described above,plasmids available for the knockout of FAR1, SST2, and HIS3, and mutantstrains that are commonly available in the research community. Thefollowing haploid strains will be constructed, using one-step ortwo-step knockout protocols described in Meth. Enzymol 194:281-301,1991: 1. MATα ste3::STE2::ste3 far1 sst2 FUS1::HIS32. MATα ste2::STE3::ste2 far1 sst2 FUS1::HIS33. MATα ste3::STE2::ste3 far1 sst2 mfα1 mfα2 FUS1::HIS34. MATa ste2::STE3::ste2 far1 sst2 mfa1 mfa2 FUS1::HIS35. MATa bar1 far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his36. MATa mfa1 mfa2 far1-1 his3::fus1-HIS3 ste2-STE3 ura3 met1 ade1 leu2

[0330] Strains 1 and 2 will be tested for their ability to grow onhistidine-deficient media as a result of autocrine stimulation of theirpheromone response pathways by the pheromones which they secrete. Ifthese tests prove successful, strain I will be modified to inactivateendogenous MFα1 and MFα2. The resulting strain 3, MATα far1 sst2ste3::STE2::ste3 FUS1::HIS3 mfa1 mfa2, should no longer display theselectable phenotype (i.e., the strain should be auxotrophic forhistidine). Similarly, strain 2 will be modified to inactivateendogenous MFa1 and MFa2. The resulting strain 4, MATa far1 sst2ste2::STE3::ste2 FUS1::HIS3 mfa1 mfa2, should be auxotrophic forhistidine. The uses of strains 5 and 6 are outlined in Examples 3 and 4below.

EXAMPLE 3 Peptide Library

[0331] In this example, a synthetic oligonucleotide encoding a peptideis expressed so that the peptide is secreted or transported into theperiplasm.

[0332] i. The region of MFα1 which encodes mature cc-factor has beenreplaced via single-stranded mutagenesis with restriction sites that canaccept oligonucleotides with AflII and BglII ends. Insertion ofoligonucleotides with AflII and BglII ends will yield plasmids whichencode proteins containing the MFα1 signal and leader sequences upstreamof the sequence encoded by the oligonucleotides. The MFα1 signal andleader sequences should direct the processing of these precursorproteins through the pathway normally used for the transport of matureα-factor.

[0333] The MFα1 gene, obtained as a 1.8 kb EcoRI fragment from pDA6300(J. Thorner, Univ. of California) was cloned into pALTER in preparationfor oligonucleotide-directed mutagenesis to remove the coding region ofmature α-factor while constructing sites for acceptance ofoligonucleotides with AflII and BclI ends. The mutagenesis wasaccomplished using the minus strand as template and the followingmutagenic oligonucleotide:5′-CTAAAGAAGAAGGGGTATCTTTGCTTAAGCTCGAGATCTCGACTGA- TAACAACAGTGTAG-3′

[0334] A HindIII site was simultaneously introduced 7 nts upstream ofthe MFα1 start codon with the oligonucleotide:5′CATACACAATATAAAGCTTTAAAAGAATGAG-3′

[0335] The resulting plasmid, Cadus 1214, contains a HindIII site 7 ntsupstream of the MFα1 initiation codon, an AflII site at the positionswhich encode the KEX2 processing site in the MFα1 leader peptide, andXhoI and BglII sites in place of all sequences from the leader-encodingsequences up to and including the normal stop codon. The 1.5 kb HindIIIfragment of Cadus 1214 therefore provides a cloning site foroligonucleotides to be expressed in yeast and secreted through thepathway normally travelled by endogenous α-factor.

[0336] A sequence comprising the ADC1 promoter and 5′ flanking sequencewas obtained as a 1.5 kb BamHI-HindIII fragment from pAAH5 (Ammerer, G.(1983) Academic Press, Inc., Meth. Enzymol. 101, 192-201 and ligatedinto the high copy yeast plasmid pRS426 (Christianson, T. W et al.(1992) Gene 110:119-122) (see FIG. 1). The unique XhoI site in theresulting plasmid was eliminated to yield Cadus 1186. The 1.5 Kb HindIIIfragment of Cadus 1214 was inserted into HindIII-digested Cadus 1186;expression of sequences cloned into this cassette initiates from theADH1 promoter. The resulting plasmid, designated Cadus 1215, can beprepared to accept oligonucleotides with AflII and BclI ends bydigestion with those restriction endonucleases. The oligonucleotideswill be expressed in the context of MFα1 signal and leader peptides(FIG. 2).

[0337] Modified versions of Cadus 1215 were also constructed. To 30improve the efficiency of ligation of oligonucleotides into theexpression vector, Cadus 1215 was restricted with KpnI and religated toyield Cadus 1337. This resulted in removal of one of two HindlIl sites.Cadus 1337 was linearized with HindIII, filled-in, and recircularized togenerate Cadus 1338. To further tailor the vector for libraryconstruction, the following double-stranded oligonucleotide was clonedinto AflII-and BglII-digested Cadus 1338: 5′TTAAGCGTGAGGCAGAAGCTTATCGATA oligo 062 3′ CGCACTCCGTCTTCGAATAGCTATCTAGoligo 063

[0338] The ClaI site is unique in the resulting vector, Cadus 1373. InCadus 1373, the HindlIl site that exists at the junction between the MFαpro sequence and the mature peptide to be expressed by this vector wasmade unique. Therefore the HindIII site and the downstream BglII sitecan be used to insert oligo-nucleotides encoding peptides of interest.These modifications of Cadus 1215 provide an laternative to the use ofthe AflII site in the cloning of oligonucleotides into the expressionsvector.

[0339] Cadus 1373 was altered further to permit elimination fromrestricted vector preparations of contaminating singly-cut plasmid. Suchcontamination could result in unacceptably high backgroundtransformation. To eliminate this possibility, approximately 1.1 kb ofdispensable ADH1 sequence at the 5′ side of the promoter region wasdeleted. This was accomplished by restruction of Cadus 1373 with SphIand BamHI, fill-in, and ligation; this maneuver regenerates the BamHIsite. The resulting vector, Cadus 1624, was then restricted with HindIIIand ClaI and an approximately 1.4 kb HindIII and ClaI fragment encoding25 lacZ was inserted to generate Cadus 1625. Use of HindIII- andBglII-restricted Cadus 1625 for acceptance of oligonucleotides resultsin a low background upon transformation of the ligation product intobacteria.

[0340] Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BclI, can be ligated into the polylinker of theexpression vector, Cadus 1215. The two single-stranded oligonucleotideshave the following sequences: 5′-G CTA CTT AAG CGT GAG GCA GAA GCT3′ and 5′-C GGA TGA TCA (NNN)_(n) AGC TTC TGC CTC ACG CTT AAG TAG C 3′

[0341] where N is any chosen nucleotide and n is any chosen integer.Yeast transformed with the resulting plasmids will secrete—through theα-factor secretory pathway—peptides whose amino acid sequence isdetermined by the particular choice of N and n). Alternatively, thefollowing single stranded oligonucleotides are used:

[0342] MFαNNK (76 mer):5′CTGGATGCGAAGACAGCTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNK      NNKNNKTGATCAGTCTGTGACGC 3′ and MFαMbo (17 mer): 5′GCGTCACAGACTGATCA 3′

[0343] When annealed the double stranded region is: TGATCAGTCTGTGACGCACTAGTCAGACACTGCG

[0344] After fill-in using Taq DNA polymerase (Promega Corporation,Madison, Wis.), the double stranded product is restricted with BbsI andMboI and ligated to HindIII- and BglII-restricted Cadus 1373.

[0345] ii. The region of MFa1 which encodes mature a-factor will bereplaced via single stranded mutagenesis with restriction sites that canaccept oligonucleotides with XhoI and AflII ends. Insertion ofoligonucleotides with XhoI and AflII ends will yield plasmids whichencode proteins containing the MFa1 leader sequences upstream of thesequence encoded by the oligonucleotides. The MFa1 leader sequencesshould direct the processing of these precursor proteins through thepathway normally used for the transport of mature a-factor.

[0346] MFA1, obtained as a BamHI fragment from pKK1 (provided by J. 30Thorner and K. Kuchler), was ligated into the BamHI site of pALTER(Promega). Using the minus strand of MFA1 as template, a HindIII sitewas inserted by oligonucleotide-directed mutagenesis just 5′ to the MFAIstart codon using the following oligonucleotide:5′CCAAAATAAGTACAAAGCTTTCGAATAGAAATGCAACCATC

[0347] A second oligonucleotide was used simultaneously to introduce ashort polylinker for later cloning of synthetic oligonucleotides inplace of MFA1 sequences. These MFA1 sequences encode the C-terminal 5amino acids of the 21 amino acid leader peptide through to the stopcodon: 5′ GCCGCTCCAAAAGAAAAGACCTCGAGCTCGCTTAAGTTCTGCGTACAAAAACGTTGTTC 3′

[0348] The 1.6 kb HindIII fragment of the resulting plasmid, Cadus 1172,contains sequences encoding the MFA1 start codon and the N-terminal 16amino acids of the leader peptide, followed by a short polylinkercontaining XhoI, SacI, and AfIII sites for insertion ofoligonucleotides. The 1.6 kb HindIII fragment of Cadus 1172 was ligatedinto HindIII-digested Cadus 1186 (see above) to place expression ofsequences cloned into this cassette under the control of the ADHIpromoter. The SacI site in the polylinker was made unique by eliminatinga second SacI site present in the vector. The resulting plasmid,designated Cadus 1239, can be prepared to accept oligonucleotides withXhoI and AfIII ends by digestion with those restriction endonucleasesfor expression in the context of MFa1 leader peptides (FIG. 3).

[0349] Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BglII, can be cloned into the polylinker of theexpression vector, Cadus 1239. The two single-stranded oligonucleotidesused for the cloning have the following sequences: 5′ GG TAC TCG AGT GAAAAG AAG GAC AAC 3′ 5′ CG TAC TTA AGC AAT AAC ACA (NNN)_(n) GTT GTC CTTCTT TTC ACT CGA GTA CC 3′

[0350] where N is any chosen nucleotide and n is any chosen integer.

[0351] Yeast transformed with the resulting plasmids willtransport—through the pathway normally used for the export ofa-factor—famesylated, carboxymethylated peptides whose amino acidsequence is determined by the particular choice of N and n (FIG. 3).

EXAMPLE 4 Peptide Secretion/Transport

[0352] This example demonstrates the ability to engineer yeast such thatthey secrete or transport oligonucleotide-encoded peptides (in this casetheir pheromones) through the pathways normally used for the secretionor transport of endogenous pheromones.

[0353] Autocrine MATa Strain CY588:

[0354] A MATa strain designed for the expression of peptides in thecontext of MFα1 (i.e., using the MFα1 expression vector, Cadus 1215) hasbeen constructed. The genotype of this strain, which we designate CY588,is MATa bar1 far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3. The bar1mutation eliminates the strain's ability to produce a protease thatdegrades α-factor and that may degrade some peptides encoded by thecloned oligonucleotides; the far1 mutation abrogates the arrest ofgrowth which normally follows stimulation of the pheromone responsepathway; an integrated FUS1-HIS3 hybrid gene provides a selectablesignal of activation of the pheromone response pathway; and, finally,the ste14 mutation lowers background of the FUS1-HIS3 readout. Theenzymes responsible for processing of the MFa1 precursor in MATα cellsare also expressed in MATa cells (Sprague and Thomer, in The Molecularand Cellular Biology of the Yeast Saccharomyces: Gene Expression, 1992,Cold Spring Harbor Press), therefore, CY588 cells should be able tosecrete peptides encoded by oligonucleotides expressed from plasmidCadus 1215.

[0355] A high transforming version (tbtl-1) of CY588 was obtained bycrossing CY1013 (CY588 containing an episomal copy of the STE14 gene)(MATa barl::hisGfar1-1 fusl-HIS3 ste14::TRP1 ura3 trpl leu2 his3 [STE14URA3 CEN4) to CY793 (MATα˜ tbtl-1 ura3 leu2 trpl his3 fusl-HIS2 can1ste114::TRP1 [FUS1 LEU2 2μ] and selecting from the resultant spores astrain possessing the same salient genotype described for CY588 (seeabove), and in addition the tbl-1 allele, which confers the capacity forvery high efficiency transformation by electroporation. The selectedstrain is CY1455 (MA Tabarl::hisGfar1-1 fusl-HIS3 ste14:: TRP1 tbt-1ura3 trpl leu2 his3).

[0356] Secretion of Peptides in the Context of Yeast α-Factor:

[0357] Experiments were performed to test: 1. the ability of Cadus 1215to function as a vector for the expression of peptides encoded bysynthetic oligonucleotides; 2. the suitability of the oligonucleotides,as designed, to direct the secretion of peptides through the α-factorsecretory pathway; 3. the capacity of CY588 to secrete those peptides;and 4. the ability of CY588 to respond to those peptides that stimulatethe pheromone response pathway by growing on selective media. Theseexperiments were performed using an oligonucleotide which encodes the 13amino acid α-factor; i.e., the degenerate sequence (NNN)_(n) in theoligonucleotide cloned into Cadus 1215 (see above) was specified (n=13)to encode this pheromone. CY588 was transformed with the resultingplasmid (Cadus 1219), and transformants selected on uracil-deficientmedium were transferred to histidine-deficient medium supplemented witha range of concentrations of aminotriazole (an inhibitor of the HIS3gene product that serves to reduce background growth). The resultsdemonstrate that the synthetic oligo-nucleotide, expressed in thecontext of MFα1 by Cadus 1215, conferred upon CY588 an ability to growon histidine-deficient media supplemented with aminotriazole. Insummation, these data indicate that: 1. CY588 is competent for thesecretion of a peptide encoded by the (NNN)_(n) sequence of thesynthetic oligonucleotide cloned into and expressed from Cadus 1215; and2. CY588 can, in an autocrine fashion, respond to a secreted peptidewhich stimulates its pheromone response pathway, in this case byα-factor binding to STE2.

[0358] Additional experiments were performed to test the utility ofautocrine yeast strains in identifying agonists of the Ste2 receptorfrom among members of two semi-random α-factor libraries, α-Mid-5 andMFα-8.

[0359] α-Mid-5 Library

[0360] A library of semi-random peptides, termed the α-Mid-5 library,was constructed. In this library, the N-terminal four amino acids andthe C-terminal four amino acids of a 13 residue peptide are identical tothose of native α-factor while the central five residues (residues 5-9)are encoded by the degenerate sequence (NNQ)₅. The followingoligonucleotides were used in the construction of the α-Mid-5 library:(1) MFαMbo, a 17 mer: 5′GCGTCACAGACTGATCA (2) MID5ALF, a 71 mer: 5′GCCGTCAGTAAAGCTTGGCATTGGTTGNNQNNQNNQNNQMMQCAGCCTAT GTACTGATCAGTCTGTGACGC

[0361] Sequenase (United States Biochemical Corporation, Cleveland,Ohio) was used to complete the duplex formed after annealing MFαMbo tothe MID5ALF oligonucleotide. In the MID5ALF sequence, N indicates amixture of A, C, G, and T at ratios of 0.8:1:1.3:1; Q indicates amixture of C and G at a ratio of 1:1.3. These ratios were employed tocompensate for the different coupling efficiences of the bases duringoligonucleotide synthesis and were thus intended to normalize theappearance of all bases in the library. The double-strandedoligonucleotide was restricted with HindIII and MboI and ligated toCadus 1625 (see above); Cadus 1625 had been prepared to accept thesemi-random oligonucleotides by restriction with HindIII and BgIII.

[0362] The apparent complexity of the αMid-5 library is 1×10⁷. Thiscomplexity is based on the number of bacterial transformants obtainedwith the library DNA versus transformants obtained with control vectorDNA that lacks insert. Sequence analysis of six clones from the librarydemonstrated that each contained a unique insert.

[0363] To identify peptide members of the α-mid-5 library that could actas agonists on the STE2 receptor, CY1455, a high transforming version ofCY588, was electroporated to enhance uptake of α-Mid-5 DNA.Transformants were selected on uracil-deficient (-Ura) syntheticcomplete medium and were transferred to histidine-deficient (-His)synthetic complete medium supplemented with 0.5 mM or 1 mMaminotriazole.

[0364] Yeast able to grow on -His+aminotriazole medium include (1) yeastwhich are dependent on the expression of an α-factor variant agonist and(2) yeast which contain mutations that result in constitutive signallingalong the pheromone pathway. Yeast expressing and secreting a variantSTE2 receptor agonist have the ability to stimulate the growth on -Hismedium of surrounding CY 1455 cells that do not express such an agonist.Thus a recognizable formation (termed a “starburst”) results, consistingof a central colony, growing by virtue of autocrine stimuation of thepheromone pathway, surrounded by satellite colonies, growing by virtueof paracrine stimulation of the pheromone pathway by the agonist peptideas that peptide diffuses radially from the central, secreting colony.

[0365] In order to identify the peptide sequence responsible for this“starburst” phenomenon, yeast were transferred from the center of the“starburst” and streaks were made on -Ura medium to obtain singlecolonies. Individual clones from -Ura were tested for the His+ phenotypeon -His+ aminotriazole plates containing a sparse lawn of CY1455 cells.Autocrine yeast expressing a peptide agonist exhibited the “starburst”phenotype as the secreted agonist stimulated the growth of surroundingcells that lacked the peptide but were capable of responding to it.Constitutive pheromone pathway mutants were capable of growth on-His+aminotriazole but were incapable of enabling the growth ofsurrounding lawn cells.

[0366] Alternatively, streaks of candidate autocrine yeast clones weremade on plates containing 5-fluoroorotic acid (FOA) to obtain Urasegregants were retested on -His+ aminotriazole for the loss of theHis+phenotype. Clones that lost the ability to grow on -His+aminotriazole after selection on FOA (and loss of the peptide-encodingplasmid) derived from candidate expressors of a peptide agonist. Theplasmid was rescued from candidate clones and the peptide sequencesdetermined. In addition, a plasmid encoding a putative Ste2 agonist wasreintroduced into CY1455 to confirm that the presence of the plasmidencoding the peptide agonist conferred the His+phenotype to CY1455.

[0367] By following the above protocol novel Ste2 agonists have beenidentified from the α-Mid-5 library. Sequences of nine agonists follow,preceded by the sequence fo the native α-factor pheromone and by theoligonucleotide used to encode the native pheromone in theseexperiments. (Note the variant codons used in the α-factor-encodingoligonucleotide for glutamine and proline in the C-terminal amino acidsof α-factor). α-factor TGG CAT TGG TTG CAG CTA AAA CCT GGC CAAA CCA ATGTAC encodes Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr α-factoroligo: TGG CAT TGG TTG CAG CTA AAA CCT GGC CAG CCT ATG TAC encodes TrpHis Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr M1 TGG CAT TGG TTG TCCTTG TCG CCC GGG CAG CCT ATG TAC encodes Trp His Trp Leu Ser Leu Ser ProGly Gln Pro Met Tyr M2 TGG CAT TGG TTG TCC CTG GAC GCT GGC CAG CCT ATGTAC encodes Trp His Trp Leu Ser Leu Asp Ala Gly Gln Pro Met Tyr M3 TGGCAT TGG TTG ACC TTG ATG GCC GGG CAG CCT ATG TAC encodes Trp His Trp LeuThr Leu Met Ala Gly Gln Pro Met Tyr M4 TGG CAT TGG TTG CAG CTG TCG GCGGGC CAG CCT ATG TAC encodes Trp His Trp Leu Gln Leu Ser Ala Gly Gln ProMet Tyr M5 TGG CAT TGG TTG AGG TTG CAG TCC GGC CAG CCT ATG TAC encodesTrp His Trp Leu Arg Leu Gln Ser Gly Gln Pro Met Tyr M6 TGG CAT TGG TTGCGC TTG TCC GCC GGG CAG CCT ATG TAC encodes Trp His Trp Leu Arg Leu GlnSer Gly Gln Pro Met Tyr M7 TGG CAT TGG TTG TCG CTC GTC CCG GGG CAG CCTATG TAC encodes Trp His Trp Leu Ser Leu Val Pro Gly Gln Pro Met Tyr M8TGG CAT TGG TTG TCC CTG TAC CCC GGG CAG CCT ATG TAC encodes Trp His TrpLeu Ser Leu Tyr Pro Gly Gln Pro Met Tyr M9 TGG CAT TGG TTG CGG CTG CAGCCC GGG CAG CCT ATG TAC encodes Trp His Trp Leu Arg Leu Gln Pro Gly GlnPro Met Tyr

[0368] The nine peptide agonists of the Ste2 receptor above were derivedfrom one electroporation of CY1455 using 1 μg of the α-Mid-5 libraryDNA. Approximately 3×10⁵ transformants were obtained, representingapproximately 0.03% of the sequences present in that library.

[0369] MFα-8 Library

[0370] A semi-random α-factor library was obtained through synthesis ofmutagenized α-factor oligonucleotides such that 1 in 10,000 peptideproducts were expected to be genuine α-factor. The mutagenesis wasaccomplished with doped synthesis of the oligonucleotides: eachnucleotide was made approximately 68% accurate by synthesizing thefollowing two oligos: 5′ CTGGATG CGAAGACTCAGCT (20 mer) (oligo060) 5′CGGATGATCA gta cat tgg ttg gcc agg ttt tag ctg caa cca atg cca AGC TGAGTC TTC GCATCC-AG (69 mer) (oligo074)

[0371] The lower case letters indicate a mixture of 67% of thatnucleotide and 11% of each of the other three nucleotides (e.g. tindicates 67% T and 11% A, 11% C, and 11% G). Note that digestion of thedouble-stranded oligo-nucleotide by FokI or BbsI will yield an identical5′ end that is compatible with HindIII ends.

[0372] Oligos 060 and 074 will form the following double-strandedmolecule when annealed: 5′-CTGGATGCGAAGACTCAGCT 3′-GACCTAC-GCTTCTGAGTCGAacc gta acc aac gtc gat ttt gga ccg gtt ggt tac atg ACTAGTAGGC-5′

[0373] The duplex was repetitively filled-in using Taq DNA polymerase(Promega Corporation, Madison, Wis.). The double-stranded product wasrestricted with BbsI and BclI and ligated into HindIII- andBglII-digested Cadus 1373. The BglII/BclI joint creates a TGA stop codonfor the termination of translation of the randomers. Using thisapproach, the MFα-5.8 library (a library of apparent low complexitybased on PCR analysis of oligonucleotide insert frequency) wasconstructed.

[0374] To identify peptide members of the MFα-5.8 library that could actas agonists on the STE2 receptor, CY1455, a high transforming version ofCY588, was electroporated to enhance uptake of MFα-5.8 DNA.Transformants were selected on uracil-deficient (-Ura) syntheticcomplete medium and were transferred to histidine-deficient (-His)synthetic complete medium supplemented with 1.0 mM or 2.5 mMaminotriazole. Yeast from colonies which were surrounded by satellitegrowth were transferred as streaks to -Ura medium to obtain singlecolonies. Yeast from single colonies wree then tested for the His+phenotype on -His+ aminotriazole plates. Sequence analysis of seven ofthe plasmids rescued from His+ yeast revealed three unique α-factorvariants that acted as agonists on the STE2 receptor.

[0375] 1.4 independent clones had the following sequence: TGG CAT TGGCTA CAG CTA ACG CCT GGG CAA CCA ATG TAC encoding Trp His Trp Leu Gln LeuThr Pro Gly Gln Pro Met Tyr

[0376] 2.2 independent clones had the following sequence: TGG CAT TGGCTG GAG CTT ATG CCT GGC CAA CCA TTA TAC encoding Trp His Trp Leu Glu LeuMet Pro Gly Gln Pro Leu Tyr 3. TGG CAT TGG ATG GAG CTA AGA CCT GGC CAACCA ATG TAC encoding Trp His Trp Met Glu Leu Arg Pro Gly Gln Pro Met Tyr

[0377] Autocrine Mata Strain CY599:

[0378] A MATa strain designed for the expression of peptides in thecontext of MFA1 (i.e., using the MFA1 expression vector, Cadus 1239) hasbeen constructed. The genotype of this strain, designated CY599, is MATamfa1 mfa2 far1-1 his3::fusl-HIS3 ste2-STE3 ura3 metl adel leu2. In thisstrain, Cadus 1147 (see above) was used to replace STE2 with a hybridgene in which the STE3 coding region is under the control of expressionelements which normally drive the expression of STE2. As a result, thea-factor receptor replaces the α-factor receptor. The genes which encodea-factor are deleted from this strain; the far1 mutation abrogates thearrest of growth which normally follows stimulation of the pheromoneresponse pathway; and the FUS1-HIS3 hybrid gene (integrated at the HIS3locus) provides a selectable signal of activation of the pheromoneresponse pathway. CY599 cells were expected to be capable of thetransport of a-factor or a-factor-like peptides encoded byoligonucleotides expressed from Cadus 1239 by virtue of expression ofthe endogenous yeast transporter, Ste6.

[0379] Transport of Peptides by the Yeast a-Factor Pathway:

[0380] Experiments were performed to test: 1. the ability of Cadus 1239to function as a vector for the expression of peptides encoded bysynthetic oligonucleotides; 2. the suitability of the oligonucleotides,as designed, to direct the export of famesylated, carboxymethylatedpeptides through the pathway normally used by a-factor; 3. the capacityof CY599 to export these peptides; and 4. the ability of CY599 torespond to those peptides that stimulate the pheromone response pathwayby growing on selective media. These tests were performed using anoligonucleotide which encodes the 12 amino acid a-factor; specifically,the degenerate sequence (NNN)_(n) in the oligo-nucleotide cloned intoCadus 1239 (see above) (with n=12) encodes the peptide component ofa-factor pheromone. CY599 was transformed with the resulting plasmid(Cadus 1220), and transformants selected on uracil-deficient medium weretransferred to histidine-deficient medium supplemented with a range ofconcentrations of aminotriazole. The results demonstrate that thesynthetic oligonucleotide, expressed in the context of MFA1 by Cadus1220, conferred upon CY599 enhanced aminotriazole-resistant growth onhistidine-deficient media. In summation, these data indicate: 1. Cadus1220 and the designed oligonucleotide are competent to direct theexpression and export of a famesylated, carboxymethylated peptideencoded by the (NNN)_(n) sequence of the synthetic oligonucleotide; and2. CY599 can, in an autocrine fashion, respond to a famesylated,carboxymethylated peptide that stimulates its pheromone responsepathway, in this case signaling initiates as a-factor binds to STE3.

EXAMPLE 5 Proof of Concept

[0381] This example will demonstrate the utility of the autocrine systemfor the discovery of peptides which behave as functional pheromoneanalogues. By analogy, this system can be used to discover peptides thatproductively interact with any pheromone receptor surrogates.

[0382] CY588 (see strain 5, Example 2 above) will be transformed withCADUS 1215 containing oligonucleotides encoding random tridecapeptidesfor the isolation of functional α-factor analogues. CYS99 (see strain 6,Example 2 above) will be transformed with CADUS 1239 containing oligosof random sequence for the isolation of functional a-factor analogues.Colonies of either strain which can grow on histidine-deficient mediafollowing transformation will be expanded for the preparation of plasmidDNA, and the oligo-nucleotide cloned into the expression plasmid will besequenced to determine the amino acid sequence of the peptide whichpresumably activates the pheromone receptor. This plasmid will then betransfected into an isogenic strain to confirm its ability to encode apeptide which activates the pheromone receptor. Successful completion ofthese experiments will demonstrate the potential of the system for thediscovery of peptides which can activate membrane receptors coupled tothe pheromone response pathway.

[0383] Random oligonucleotides to be expressed by the expression plasmidCADUS 1215 will encode tridecapeptides constructed as 5′CGTGAAGCTTAAGCGTGAGGCAGAAGCT(NNK)₁₃ TGATCATCCG, where N is anynucleotide, K is either T or G at a ratio of 40:60 (see Proc. Natl.Acad. Sci. 87:6378, 1990; ibid 89:5393, 1992), and the AfilI and Bellsites are underlined. This oligonucleotide is designed such that: theAflII and Bell sites permit inserting the oligos into the AflII andBglII site of CADUS 1215, the HindIII site just 5′ to the AflII site inthe 5′ end of the oligo allows future flexibility with cloning of theoligos; the virtual repeat of GAGGCT and the GAGA repeats which arepresent in the wild-type sequence and which can form triple helixes arechanged without altering the encoded amino acids. The randomoligonucleotides described above will actually be constructed from thefollowing two oligos: 5′ CGTGAAGCTTAAGCGTGAGGCAGAAGCT and 5′CGGATGATCA(MNN)₁₃AGCTTCTG,

[0384] where M is either A or C at a ratio of 40:60. The oligos will beannealed with one another and repetitively filled in, denatured, andreannealed (Kay et al, Gene, 1993). The double-stranded product will becut with AflII and Bell and ligated into the AflII- and BglII-digestedCADUS 1215. The BglII/BclI joint will create a TGA stop codon fortermination of translation of the randomers. Because of the TA contentof the Afl overhang, the oligos will be ligated to the AflII-andBglII-digested pADC-MFα at 4° C.

[0385] Random oligonucleotides to be expressed by the expression plasmidCADUS 1239 will encode monodecapeptides constructed as5′ GGTACTCGAGTGAAAAGAAGGACAAC(NNK)₁₁TGTGTTATTGCTTAAGTACG,

[0386] where N is any nucleotide, K is either T or G at a ratio of 40:60(see Proc. Natl. Acad. set 87:6378, 1990; ibid 89:5393, 1992). Whencloned into the XhoI and AflII sites of CADUS 1239 the propeptidesexpressed under the control of the ADH1 promoter will contain the entireleader peptide of MFa1, followed by 11 random amino acids, followed bytriplets encoding CVIA (the C-terminal tetrapeptide of wild-typea-factor). Processing of the propeptide should result in the secretionof dodecapeptides which contain 11 random amino acids followed by aC-terminal, famesylated, carboxymethylated cysteine.

[0387] Using the procedure described above, the oligonucleotides forexpression in CADUS 1239 will actually be constructed from the followingtwo oligos: 5′ GGTACTCGAGTGAAAAGAAGGACAAC and5′ CGTACTTAAGCAATAACACa(MNN)₁₁GTTGTCC,

[0388] where M is either A or C at a ratio of 40:60, and the XhoI andAflII sites are underlined.

[0389] Discovery of a-Factor Analoques from a Random Peptide Library

[0390] An optimized version of strain 6 (Example 2 above) was derived.This yeast strain, CY2012 (MATa ste2-STE3far1 Δ1442 mfal::LEU2mfa2-lacZfusl-HIS3 tbtl-1 ura3 leu2 his3 trpl suc2), was constructed asfollows. From a cross of CY570 (MATa mfal::LEU2 mfa2-lacZ ura3 trplhis3Δ200 can1 leu2fusl-HIS3 [MFA1 URA3 2μ] [Fus1Δ8-73 TRP1 CEN6]) byCY1624 (MATα tbtl-1 fus1-HIS3 trpl ura3 leu2 his3 lys2-801 SUC+), aspore was selected (CY1877) of the following genotype: MATa mfa1::LEU2mfa2-lacZ fus1-HIS3 tbtl-1 ura3 leu2 his3 trp1 suc2. This strain lacksboth genes (NFA1 and MFA2) encoding a-factor precursors, contains theappropriate pheromone pathway reporter gene (fusl-HIS3), and transformsby electroporation at high efficiency (tbtl-l). This strain was alteredby deletion of the FAR1 gene (with Cadus 1442; see Example 6), andreplacement of STE2 coding sequences with that of STE3 (see Example 1)to yield CY2012.

[0391] This strain was transformed with plasmid DNA from a randoma-factor library by electroporation and plated on 17 synthetic completeplates lacking uracil (-Ura), yielding approximately 10⁵ Ura+ coloniesper plate after 2 days at 30° C. These colonies were replica plated tohistidine-deficient synthetic complete media (-His) containing 0.2 mM3-aminotriazole and after three days at 30° C. 35 His+ replicas werestreaked to -Ura plates. The resultant colonies, 3 from each isolate,were retested for their His+ phenotype, and streaked to 5-fluorooroticacid plates to obtain Ura segregants (lacking a library plasmid). ThoseUra-segregants were tested for the loss of their His+ phenotype. Ten ofthe original isolates passed these tests; in two cases only one of thethree Ura+ colonies purified from the isolate retained the His+phenotype, but nevertheless subsequently segregated Ura His− colonies.

[0392] A single plasmid (corresponding to a bacterial colony) wasobtained from each of the ten isolates, and reintroduced into CY2012.Eight of the ten plasmids passed the test of retaining the ability toconfer the His+ phenotype on CY2012 (the two that failed correspond tothe two isolates that were mentioned above, suggesting that theseisolates contain at least one “irrelevant’ plasmid). Sequencing of therandomized insert in the eight plasmids of interest revealed that fourcontain the sequence: TAT GCT CTG TTT GTT CAT TTT TTT GAT ATT CCG TyrAla Leu Phe Val His Phe Phe Asp Ile Pro

[0393] two contain the sequence: TTT AAG GGT CAG GTG CGT TTT GTG GTT CTTGCT Phe Lys Gly Gln Val Arg Phe Val Val Leu Ala,

[0394] and two contain the sequence: CTT ATG TCT CCG TCT TTT TTT TTT TTGCCT GCG Leu Met Ser Pro Ser Phe Phe Phe Leu Pro Ala

[0395] Clearly, these sequences encode novel peptides, as the nativea-factor sequence differs considerably: Tyr Ile Ile Lys Gly Val Phe TrpAsp Pro Ala.

[0396] The a-factor variants identified from random peptide librarieshave utility as “improved” substrates of ABC transporters expressed inyeast. For example, identification of a preferred substrate of humanMDR, one that retains agonist activity on the pheromone receptor, wouldpermit the establishment of robust yeast screens to be used in thediscovery of compounds that affect transporter function.

EXAMPLE 6 Functional Expression of a Mammalian G Protein-CoupledReceptor and Ligand in an Autocrine Yeast Strain

[0397] This example details the following: (1) expression of human C5areceptor in yeast; (2) expression of the native ligand of this receptor,human C5a, in yeast; and (3) activation of the endogenous yeastpheromone pathway upon stimulation of the C5a receptor by C5a when bothof these molecules are expressed within the same strain of autocrineyeast. Following the experimental data we outline the utility ofautocrine strains of yeast that functionally express the human C5areceptor.

[0398] Human C5a is a 74 amino acid polypeptide that derives from thefifth component of complement during activation of the complementcascade; it is the most potent of the complement-derived anaphylatoxins.C5a is a powerful activator of neutrophils and macrophage functionsincluding production of cytotoxic super oxide radicals and induction ofchemotaxis and adhesiveness. In addition C5a stimulates smooth musclecontraction, induces degranulation of mast cells, induces serotoninrelease from platelets and increases vascular permeability. The C5aanaphylatoxin can also amplify the inflammatory response by stimulatingthe production of cytokines. As C5a is a highly potent inflammatoryagent, it is a primary target for the development of antagonists to beused for intervention in a variety of inflammatory processes.

[0399] The C5a receptor is present on neutrophils, mast cells,macrophages and smooth muscle cells and couples through G proteins totransmit signals initiated through the binding of C5a.

[0400] Expression of the C5a Receptor

[0401] The plasmid pCDM8-C5aRc, bearing cDNA sequence encoding the humanC5a receptor, was obtained from N. Gerard and C. Gerard (Harvard MedicalSchool, Boston, MA) (Gerard and Gerard 1991). Sequence encoding C5a wasderived from this plasmid by PCR using VENT polymerase (New EnglandBiolabs Inc., Beverly Mass.), and the following primers:#1-GGTGGGAGGGTGCTC T CTAGAAGGAAGTGTTCACC #2-GCCCAGGAGACCAGA C C ATGGACTCCTTCAATTATACCACC

[0402] Primer #1 contains a single base-pair mismatch (underlined) toC5a receptor cDNA. It introduces an XbaI site (in bold) 201 bpdownstream from the TAG termination codon of the C5a receptor codingsequence. Primer #2 contains two mismatched bases and serves to createan NcoI site (in bold) surrounding the ATG initiator codon (doubleunderlined). The second amino acid is changed from an aspartic acid toan asparagine residue. This is the only change in primary amino acidsequence from the wild type human C5a receptor.

[0403] The PCR product was restricted with NcoI and XbaI (sites in bold)and cloned into CADUS 1002 (YEp51Nco), a Gal10 promoter expressionvector. The sequence of the entire insert was determined by dideoxysequencing using multiple primers. The sequence between the NcoI andXbaI sites was found to be identical to the human C5a receptor sequencethat was deposited in GenBank (accession #J05327) with the exception ofthose changes encoded by the PCR primers. The C5a receptor-encodinginsert was transferred to CADUS 1289 (pLPXt), a PGK promoter expressionvector, using the NcoI and XbaI sites, to generate the C5a receptoryeast expression clone, CADUS 1303.

[0404] A version of the C5a receptor which contains a yeast invertasesignal sequence and a myc epitope tag at its amino terminus wasexpressed in Cadus 1270-transferred yeast under control of a GAL10promoter. Plasmids encoding an untagged version of the C5a receptor anda myc-tagged derivative of FUSI served as controls. The expression ofthe tagged receptor in yeast was confirmed by Western blot using theanti-myc monoclonal antibody 9E10. In the lane containing the extractfrom the Cadus 1270-transformant, the protein that is reactive with theanti-myc monoclonal antibody 9E 10 was approximately 40 kD in size, asexpected. Note that this receptor construct is not identical to the oneused in the autocrine activation experiments. That receptor is nottagged, does not contain a signal sequence and is driven by the PGKpromoter.

[0405] Expression of the Ligand, C5a

[0406] A synthetic construct of the sequence encoding C5a was obtainedfrom C. Gerard (Harvard Medical School, Boston, Mass.). This syntheticgene had been designed as a FLAG-tagged molecule for the secretion fromE. coli (Gerard and Gerard (1990) Biochemistry 29:9274-9281). The C5acoding region, still containing E. coli codon bias, was amplified usingVENT polymerase (New England Biolabs Inc., Beverly Mass.) through 30cycles using the following primers: C5a5′= CCCCTTAAGCGTGAGGCAGAAGCTACTCTGCAAAAGAAGATC C5a3′= GAAGATCTTCAGCGGCCGAGTTGCATGTC

[0407] A PCR product of 257 bp was gel isolated, restricted with AflIIand BglII, and cloned into CADUS 1215 (an expression vector designed toexpress peptide sequences in the context of Mfoc) to yield CADUS 1297.The regions of homology to the synthetic C5a gene are underlined. The 5′primer also contains pre-pro α-factor sequence. Upon translation andprocessing of the pre-pro α-factor sequence, authentic human C5a shouldbe secreted by yeast containing CADUS 1297. The insert sequence in CADUS1297 was sequenced in both orientations by the dideoxy method and foundto be identical to that predicted by the PCR primers and the publishedsequence of the synthetic C5a gene (Franke et al. (1988) Methods inEnzymology 162: 653-668).

[0408] Two sets of experiments, aside from the autocrine activation ofyeast detailed below, demonstrated that CADUS 1297 can be used toexpress C5a in yeast.

[0409] 1). C5a was immunologically detected in both culture supernatantand lysed cells using a commercially available enzyme-linkedimmunosorbent assay (ELISA)(Table 1). This assay indicated theconcentration of C5a in the culture supernatant to be approximately 50to 100 nM. In comparison, in data derived from mammalian cells, thebinding constant of C5a to its receptor is 1 nM (Boulay et al.(1991)Biochemistry 30:2993-2999.

[0410] 2). C5a expressed in yeast was shown to compete for binding withcommercially obtained (Amersham Corporation, Arlington Heights, Ill.),radiolabeled C5a on induced HL60 cells.

[0411] Activation of the Pheromone Response Pathway in Autocrine YeastExpressing the Human C5a Receptor and Human C5a

[0412] Activation of the yeast pheromone response pathway through theinteraction of C5a with the C5a receptor was demonstrated using a growthread-out. The strain used for this analysis, CY455 (MATα :tbt1-1 ura3leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1 ste3*1156) contains thefollowing significant modifications. A pheromone inducible HIS3 gene ,fus1-HIS3, is integrated at the Fusl locus. A hybrid gene containingsequence encoding the first 41 amino acids of GPA1 (the yeast Gαsubunit) fused to sequence encoding human Gαi2a (minus codons for theN-terminal 33 amino acids) replaces GPA1 at its normal chromosomallocation. The yeast STE14 gene is disrupted to lower the basal level ofsignaling through the pheromone response pathway. The yeast a-factorreceptor gene, STE3, is deleted. The last two modifications are probablynot essential, but appear to improve the signal-to-noise ratio.

[0413] CY455 (MATα tbt1-1 ura3 leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1ste3*1156) was transformed with the following plasmids:

[0414] Cadus 1289+Cadus 1215=Receptor⁻Ligand⁻=(R−L−)

[0415] Cadus 1303+Cadus 1215=Receptor⁺Ligand⁻=R+L−

[0416] Cadus 1289+Cadus 1297=Receptor⁻Ligand⁺=(R—L+)

[0417] Cadus 1303+Cadus 1297=Receptor⁺Ligand⁺=(R+L+)

[0418] Receptor refers to the human C5a receptor.

[0419] Ligand refers to human C5a.

[0420] Three colonies were picked from each transformation and grownovernight in media lacking leucine and uracil, at pH 6.8 with 25 mMPIPES (LEU URA pH 6.8 with 25 mM PIPES). This media was made by adding0.45 ml of sterile IM KOH and 2.5 ml of sterile 1 M PIPES pH 6.8 to 100ml of standard SD LEU-URA-media. After overnight growth the pH of thismedia is usually acidified to approximately pH 5.5. Overnight cultureswere washed once with 25 mM PIPES pH 6.8 and resuspended in an equalvolume of media lacking leucine, uracil and histidine (LEU URA HIS pH6.8 with 25 mM PIPES). The optical density at 600 nm of a 1/20 dilutionof these cultures was determined and the cultures were diluted into 25mM PIPES pH 6.8 to a final OD₆₀₀ of 0.2. A volume (5 ul) of thisdilution equivalent to 10,000 cells was spotted onto selective (HIS+TRP−pH 6.8) plates. Only those strains expressing both C5a and its receptor(R+L+) show growth on the selective plates which lack histidine. Alltest strains are capable of growth on plates containing-histidine. TheR+L+ strain will grow on plates containing up to 5 mM aminotriazole, thehighest concentration tested.

[0421] For verification of pheromone pathway activation andquantification of the stimulation, the activity of the fusl promoter wasdetermined colorometrically using a fus1-lacZ fusion in a similar set ofstrains. CY878 (MATα tbt1-1 fus1-HIS3 caN1 ste14::trp1::LYS2 ste3*1156gpa1(41)-Gαi2) was used as the starting strain for these experiments.This strain is a trp1 derivative of CY455. The transformants for thisexperiment contained CADUS 1584 (pRS424-fus1-lacZ) in addition to thereceptor and ligand plasmids. Four strains were grown overnight in SDLEU URA TRP pH 6.8 with 50 mM PIPES to an OD₆₀₀ of less than 0.8. Assayof β-galactosidase activity (Guarente 1983) in these strains yields thedata shown in FIG. 4. The coupling of the C5a receptor to Gα chimeras isshown in Table 2.

[0422] Uses of the Autocrine C5a Strains:

[0423] A primary use of the autocrine C5a strains will be in thediscovery of C5a antagonists. Inhibitors of the biological function ofC5a would be expected to protect against tissue damage resulting frominflammation in a wide variety of inflammatory disease processesincluding but not limited to: respiratory distress syndrome (Duchateauet al. (1984) Am Rev Respir Dis 130:1058); (Hammerschmidt et al. (1980)Lancet 1:947), septic lung injury (Olson et al. 1985) Ann Surg 202:771),arthritis (Banerjee et al. (1989) J. Immuinol 142:2237), ischemic andpost-ischemic myocardial injury (Weisman (1990) Science 146:249);(Crawford et al. (1988) Circulation 78:1449) and bum injury (Gelfand etal. (1982) J. Clin Invest 70:1170).

[0424] The autocrine C5a system as described can be used to isolate C5aantagonists as follows:

[0425] 1. High Throughput Screens to Identify Agonists of the C5aReceptor.

[0426]FIG. 5 illustrates an exemplary set of steps for isolatingsurrogate ligands for the C5a receptor by the subject autocrine SSCL™method. As described above, yeast cells were engineered to express humanC5a receptor under conditions whereby the receptor is functionallycoupled to a fus1:his3 reporter gene construct. The cells aretransformed with a library encoding random peptides (supra) and platedon selective (His⁻) media. In the first round of screening (see FIG. 5)yeast colonies are isolated by their ability to grow on the histidinedeficient plates. In order to distinguish growth due to real receptoragonists, as opposed to revertants of the histidine auxotroph, DNA wasextracted from the colonies isolated in the first round, amplified in E.coli, and transformed back into the engineered yeast cells and plated onHis⁻ plates. High frequency of transformation or “jackpots” of cellgrowth in the the second round indicates plasmids encoding genuinereceptor agonists; individual colonies were picked, plasmid DNAisolated, amplified in E. coli, and the sequence of the surrogate liganddeduced from the DNA sequence corresponding peptide-encoding region ofeach isolated plasmid.

[0427] After sequencing and deducing the amino acid sequence of theencoded surrogate ligands identified in the histidine auxotrophy rescueassay described above, individual peptides were chemically synthesized,dissolved in DMF, and spotted on a lawn of the engineered yeast cells.As illustrated in FIG. 6, C5a receptor agonists result in growth ofcells around the areas were a peptide was spotted. FIG. 7 shows theamino acid sequence for C5a surrogate agonist peptides obtained by theabove method. Interestingly, the isolates do not show extensive sequencehomology to one and other, though several duplicate isolates were foundamongst different transformants.

[0428] Using the fus1:lacZ reporter gene construct described above,yeast cells engineered to express the human C5a receptor were stimulatedwith synthetic C5a surrogate peptides at varying concentrations. FIG. 8shows the dose response curve for various of the surrogate peptideligands using on a colorimetric lacZ readout.

[0429] The activity of the surrogate ligands were subsequently tested,as shown in FIG. 9, by contacting the mammalian cell-line HEK293, whichhas been further engineered to provide a C5a receptor coupled with aCRE:lacZ reporter gene construct, with chemically synthesized versionsof the peptides identified as C5a ligands by the autocrine methoddescribed above.

[0430] To further improve the selectivity and/or potency of the agonistsidentified by the above steps, we selected a surrogate peptide (peptide122, see FIG. 7), and created degenerate peptide libraries based on thesequence of that peptide as a starting point (e.g., a semi-randomlibrary) as follows: C5a pep122Tyr-Thr-Arg-Gly-Trp-Lys-Ala-Arg-Leu-Leu-Trp-Leu-Ile sub-libraries N-termXaa-Xaa-Xaa-Xaa-Trp-Lys-Ala-Arg-Leu-Leu-Trp-Leu-Ile mid4Tyr-Thr-Arg-Gly-Xaa-Xaa-Xaa-Xaa-Leu-Leu-Trp-Leu-Ile C-termTyr-Thr-Arg-Gly-Trp-Lys-Ala-Arg-Xaa-Xaa-Xaa-Xaa-Xaa mod1Xaa-Thr-Arg-Xaa-Trp-Lys-Xaa-Arg-Leu-Xaa-Trp-Leu-Xaa mod2Tyr-Xaa-Arg-Gly-Xaa-Lys-Ala-Xaa-Leu-Leu-Xaa-Leu-Ile mod3Tyr-Thr-Xaa-Gly-Trp-Xaa-Ala-Arg-Xaa-Leu-Trp-Xaa-Ile

[0431] Following the protocols set out above for the first generationpeptide library, the second generation peptide library was screened, andindividual clones isolated based on their ability to stimulate C5areceptor dependent transcription.

[0432] The amino acid sequence for an isolate from the second generationpeptide library, designated herein 122mod1-5, was deduced to beAsp-Thr-Arg-Ser-Trp-Lys-Leu-Arg-Leu-Leu-Trp-Leu-Ala. As illustrated byFIG. 10, the 122mod1-5 peptide was chemically synthesized and contactedwith a yeast cell engineered with a human C5a receptor and fus1:lacZreporter gene construct. The activity of the peptide (122mod1-5),determined by its ability to stimulate expression of the lacZ gene, wascompared with the original peptide (122) used to generate the secondgeneration peptide library and two other C5a receptor agonists. SeeKoneatis et al. (1994) J Immunol 153:4200.

[0433] 2. Identification of Antagonists of C5a.

[0434] In another embodiment, replacement of the fus1-HIS3 read-out withone of several negative selection schemes (fus1-URA3/FOA,fus1-GAL1/galactose or deoxygalactose, Far1 sst2 or other mutations thatrender yeast supersensitive for growth arrest) would generate a testsystem in which the presence of an antagonist would result in the growthof the assay strain. Such an approach would be applicable tohigh-throughput screening of compounds as well as to the selection ofantagonists from random peptide libraries expressed in autocrine yeast.Optimization of screens of this type would involve screening the R+L+strain at a concentration of aminotriazole which ablates growth of theR+L− strain (we are currently using 0.6 to 0.8 mM) and counterscreeningthe R+L− strain at a concentration of aminotriazole which gives anidentical growth rate (we are using 0.14 mM). In addition, the systemcould employ one of several colorometric, fluorescent orchemiluminescent readouts. Some of the genes which can be fused to thefus1 promoter for these alternate read-outs include lacZ (colorometricand fluorescent substrates), glucuronidase 20 (colorometric andfluorescent substrates), phosphatases (e.g. PHO3, PHO5, alkalinephosphatase; colorometric and chemiuminescent substrates), green protein(endogenous fluorescence), horse radish peroxidase (colorometric),luciferase (chemiluminescence).

[0435] The autocrine C5a strains have further utility as follows:

[0436] 3. In the Identification of Novel C5a Agonists from RandomPeptide Libraries Expressed in Autocrine Yeast.

[0437] Novel peptide agonists would contribute to structure/functionanalyses used to guide the rational design of C5a antagonists.

[0438] 4. In the Identification of Receptor Mutants.

[0439] Constitutively active, that is, ligand independent, receptors maybe selected from highly mutagenized populations by growth on selectivemedia. These constitutively active receptors may have utility inpermitting the mapping of the sites of interaction between the receptorand the G-protein. Identification of those sites may be important to therational design of drugs to block that interaction. In addition,receptors could be selected for an ability to be stimulated by someagonists but not others or to be resistant to antagonist. These variantreceptors would aid in mapping sites of interaction between receptor andagonist or antagonist and would therefore contribute to rational drugdesign efforts.

[0440] 5. In the Identification of Molecules that Interact with Gαi2.

[0441] Compounds or peptides which directly inhibit GDP exchange fromGαi2 would have the same effect as C5a antagonists in these assays.Additional information would distinguish inhibitors of GDP exchange fromC5a antagonists. This information could be obtained through assays thatdetermine the following:

[0442] 1. inhibition by test compounds of Gαi2 activation from otherreceptors,

[0443] 2. failure of test compounds to compete with radiolabeled C5a forbinding to the C5a receptor,

[0444] 3. failure of test compounds to inhibit the activation of otherGα subunits by C5a, and

[0445] 4. inhibition by test compounds of signalling from constitutivelyactive versions of C5a, or other, receptors.

EXAMPLE 7 Construction of Xybrid Gα Genes Construction of Two Sets ofChimeric Yeast/Mammalian Gα Genes, GPA₄₁-Gα and GPA1_(Bam)-Gα

[0446] The Gα subunit of heterotrimeric G proteins must interact withboth the βγ complex and the receptor. Since the domains of Gα requiredfor each of these interactions have not been completely defined andsince our final goal requires Gα proteins that communicate with amammalian receptor on one hand and the yeast βγ subunits on the other,we desired to derive human-yeast chimeric Gα proteins with an optimizedability to perform both functions. From the studies reported here wedetermined that inclusion of only a small portion of the amino terminusof yeast Gα is required to couple a mammalian Gα protein to the yeast βγsubunits. It was anticipated that a further benefit to using theselimited chimeras was the preservation of the entire mammalian domain ofthe Gα protein believed to be involved in receptor contact andinteraction. Thus the likelihood that these chimeras would retain theirability to interact functionally with a mammalian receptor expressed inthe same yeast cell was expected to be quite high.

[0447] Plasmid Constructions.

[0448] pRS416-GPA1 (Cadus 1069).

[0449] An XbaI-SacI fragment encoding the entire GPA1 promotor region,coding region and approximately 250 nucleotides of 3′ untranslatedregion was excised from 10 YCplac111-GPA1 (from S. Reed, ScrippsInstitute) and cloned into YEp vector pRS416 (Sikorski and Hieter,Genetics 122: 19 (1989)) cut with XbaI and SacI.

[0450] Site-Directed Mutagenesis of GPA1 (Cadus 1075,1121 and 1122).

[0451] A 1.9 kb EcoRI fragment containing the entire GPA1 coding regionand 200 nucleotides from the 5′ untranslated region was cloned intoEcoRI cut, phosphatase-treated pALTER-1 (Promega) and transformed byelectroporation (Biorad Gene Pulser) into DH5αF′ bacteria to yield Cadus1075. Recombinant phagemids were rescued with M13KO7 helper phage andsingle stranded recombinant DNA was extracted and purified according tothe manufacturer's specifications. A new NcoI site was introduced at theinitiator methionine of GPA1 by oligonucleotide directed mutagenesisusing the synthetic oligonucleotide:5′GATATATTAAGGTAGGAAACCATGGGGTGTACAGTGAG 3′.

[0452] Positive clones were selected in ampicillin and severalindependent clones were sequenced in both directions across the new NcoIsite at +1. Two clones containing the correct sequences were retained asCadus 1121 and 1122.

[0453] Construction of a GPA1-Based Expression Vector (Cadus 1127).

[0454] The vector used for expression of full length and hybridmammalian Gα proteins in yeast, Cadus 1127, was constructed in thefollowing manner. A 350 nucleotide fragment spanning the 3′ untranslatedregion of GPA1 was amplified with Taq polymerase (AmpliTaq; PerkinElmer) using the oligonucleotide primers A (5′CGAGGCTCGAGGGAACGTATAATTAAAGTAGTG 3′) and B (5′GCGCGGTACCAAGCTTCAATTCGAGATAATACCC 3′). The 350 nucleotide product waspurified by gel electrophoresis using GeneClean II (Bio101) and wascloned directly into the pCRII vector by single nucleotide overlap TAcloning (InVitrogen). Recombinant clones were characterized byrestriction enzyme mapping and by dideoxynucleotide sequencing.Recombinant clones contained a novel XhoI site 5′ to the authentic GPA1sequence and a novel KpnI site 3′ to the authentic GPA1 sequence donatedrespectively by primer A and primer B.

[0455] The NotI and SacI sites in the polylinker of Cadus 1013 (pRS414)were removed by restriction with these enzymes followed by filling inwith the Klenow fragment of DNA polymerase I and blunt end ligation toyield Cadus 1092. The 1.4 kb PstI-EcoRI 5′ fragment of GPA1 fromYCplac111-GPA1 containing the GPA1 promoter and 5′ untranslated regionof GPA1 was purified by gel electrophoresis using GeneClean (Bio101) andcloned into PstI-EcoRI restricted Cadus 1013 to yield Cadus 1087. ThePCR amplified XhoI-KpnI fragment encoding the 3′ untranslated region ofGPA1 was excised from Cadus 1089 and cloned into XhoI-KpnI restrictedCadus 1087 to yield Cadus 1092. The NotI and SacI sites in thepolylinker of Cadus 1092 were removed by restriction with these enzymes,filling in with the Klenow fragment of DNA polymerase I, and blunt endligation to yield Cadus 1110. The region of Cadus 1122 encoding theregion of GPA1 from the EcoRI site at −200 to +120 was amplified withVent DNA polymerase (New England Biolabs, Beverly, Mass.) with theprimers 5′CCCGAATCCACCAATTTCTTTACG 3′ and 5′GCGGCGTCGACGCGGCCGCGTAACAGT3′.

[0456] The amplified product, bearing an EcoRI site at its 5′ end andnovel SacI, NotI and SalI sites at its 3′ end was restricted with EcoRIand SalI, gel purified using GeneClean II (Bio101), and cloned intoEcoRI and SalI restricted Cadus 1110 to yield Cadus 1127. The DNAsequence of the vector between the EcoRI site at −200 and the KpnI siteat the 3′ end of the 3′ untranslated region was verified by restrictionenzyme mapping and dideoxynucleotide DNA sequence analysis.

[0457] PCR Amplification of GPA₄₁-Gα Proteins and Cloning into Cadus1127.

[0458] cDNA clones encoding the human G alpha subunits Gαs, Gαi2, Gαi3,and S. cerevisiae GPA1 were amplified with Vent thermostable polymerase(New England Bioloabs, Beverly, Mass.). The primer pairs used in theamplification are as follows: GαS Primer 1:5′CTGCTGGAGCTCCGCCTGCTGCTGCTGGGTGCTGGAG3′ (SacI 5′) Primer 2:5′CTGCTGGTCGACGCGGCCGCGGGGGTTCCTTCTTAGAAGCAGC3′ (Sa1I 3′) Primer 3:5′GGGCTCGAGCCTTCTTAGAGCAGCTCGTAC3′ (XhoI 3′) Gαi2 Primer 1:5′CTGCTGGAGCTCAAGTTGCTGCTGTTGGGTGCTGGGG3′ (SacI5′) Primer 2:5′CTGCTGGTCGACGCGGCCGCGCCCCTCAGAAGAGGCCGCGGTCC3′ (Sa1I 3′) Primer 3:5′GGGCTCGAGCCTCAGAAGAGGCCGCAGTC3′ (XhoI 3′) Gαi3 Primer 1:5′CTGCTGGAGCTCAAGCTGCTGCTACTCGGTGCTGGAG3′ (SacI5′) Primer 2:5′CTGCTGGTCGACGCGGCCGCCACTAACATCCATGCTTCTCAAT (Sa1I 3′) AAAGTC3′ Primer3: 5′GGGCTCGAGCATGCTTCTCAATAAAGTCCAC3′ (XhoI 3′)

[0459] After amplification, products were purified by gelelectrophoresis using GeneClean II (Bio101) and were cleaved with theappropriate restriction enzymes for cloning into Cadus 1127.

[0460] The hybrid GPA₄₁-G _(α) subunits were cloned via a SacI siteintroduced at the desired position near the 5′ end of the amplifiedgenes and a SalI or XhoI site introduced in the 3′ untranslated region.Ligation mixtures were electroporated into competent bacteria andplasmid DNA was prepared from 50 cultures of ampicillin resistantbacteria.

[0461] Construction of Integrating Vectors Encoding GPA₄₁-G _(α)Subunits. The coding region of each GPA₄₁-G _(α) hybrid was cloned intoan integrating vector (pRS406 =URA3 AmpR) using the BssHII sitesflanking the polylinker cloning sites in this plasmid. Cadus 1011(pRS406) was restricted with BssHII, treated with shrimp alkalinephosphatase as per the manufacturer's specifications, and the linearizedvector was purified by gel electrophoresis. Inserts from each of theGPA₄₁ G _(α) hybrids were excised with BssHII from the parental plasmid,and subcloned into gel purified Cadus 1011.

[0462] Construction of GPA_(BAM)-Gα Constructs.

[0463] A novel BamHI site was introduced in frame into the GPA1 codingregion by PCR amplification using Cadus 1179 (encoding a wildtype GPA1allele with a novel NcoI site at the initiator methionine) as thetemplate, VENT polymerase, and the following primers: Primer A=5′GCATCCATCAATAATCCAG 3′ and Primer B=5′ GAAACAATGGA-TCCACTTCTTAC 3′. The1.1 kb PCR product was gel purified with GeneClean II (Bio101),restricted with NcoI and BamHI and cloned into NcoI-BamHI cut andphosphatased Cadus 1122 to yield Cadus 1605. The sequence of Cadus 1605was verified by restriction analysis and dideoxy-sequencing ofdouble-stranded templates. Recombinant GPA_(Bam)-Gα hybrids of Gαs,Gαi2, and Gα16 were generated. Construction of Cadus 1855 encodingrecombinant GPA_(Bam)-Gα 16 serves as a master example: construction ofthe other hybrids followed an analogous cloning strategy. The parentalplasmid Cadus 1617, encoding native Gα16, was restricted with NcoI andBamHI, treated with shrimp alkaline phosphatase as per themanufacturer's specifications and the linearized vector was purified bygel electrophoresis. Cadus 1605 was restricted with NcoI and BamHI andthe 1.1 kb fragment encoding the amino terminal 60% of GPA1 with a novelBamHI site at the 3′ end was cloned into the NcoI- and BamHI-restrictedCadus 1617. The resulting plasmid encoding the GPA_(Bam)-Gα16 hybrid wasverified by restriction analysis and assayed in tester strains ror anability to couple to yeast Gβγ and thereby suppress the gpa1 nullphenotype. Two additional GPA_(Bam)-Gα hybrids, GPA_(Bam)-Gαs andGPA_(Bam)-Gαi2, described in this application were prepared, in ananalogous manner using Cadus1606 as the parental plasmid for theconstruction of the GPA_(Bam)-Gαi2 hybrid and Cadus 1181 as the parentalplasmid for the construction of the GPA_(Bam)-Gα s hybrid.

[0464] Coupling by Chimeric Gα Proteins.

[0465] The Gα chimeras described above were tested for the ability tocouple a mammalian G protein-coupled receptor to the pheromone responsepathway in yeast. The results of these experiments are outlined in Table3. Results obtained using GPA1₄₁-Gαi2 to couple the human C5a receptorto the pheromone response pathway in autocrine strains of yeast aredisclosed in above.

EXAMPLE 8 Screening for Modulators of G-Alpha Activity

[0466] Screens for modulators of Gα activity may also be performed asshown in the following examples for illustration purposes, which areintended to be non-limiting.

[0467] Strains CY4874 and CY4877 are isogenic but for the presence ofQ205L mutation in the cloned Gα_(i2) gene cloned into plasmid 1. StrainsCY4901 and CY4904 each have a chromosomally integrated chimeric Gαfusion comprising 41 amino acids of gpa1 at the N terminus of the humanGα_(i2) gene and are isogenic but for the presence of a constitutivelyactivating mutation in the C5a receptor gene of CY4901. Strain CY5058 isa gpa1 mutant which carries only the yeast Gβγ subunits and no Gαsubunit. This strain is a control strain to demonstrate specificity ofaction on the Gα subunit.

[0468] I. Suppression of Activation by Mutation of Gα

[0469] The Q205L mutation is a constitutively activated GTPase deficientmutant of the human Gα_(i2) gene. Antagonist compounds, chemicals orother substances which act on Gα_(i2) can be recognized by their actionto reduce the level of activation and thus reduce the signal from thefus1-lacZ reporter gene on the second plasmid (Plasmid 2).

[0470] A. GTPase gα_(i2) Mutants

[0471] test component=gpa₄₁-gα_(i2) (Q₂₀₅L)

[0472] control ocmponent=gpa₄₁-gα_(i2)

[0473] As well as the CY4874 and CY4877 constructs detailed above,similar strains with fus1-His3 or fus2-CAN-1 growth readouts may also beused. The fus1-His3 strains are preferred for screening for agonists andthe fus2-CAN1 strains are preferred for antagonist screens. test controlReadout strain effect of Gα_(i2) antagonist strain fus1-HIS3 CY4868inhibit growth of -HIS + CY4871 AT (Aminotriazole) fus1-lacZ CY4874reduce β-gal activity CY4877 fus2-CAN1 CY4892 induce growth on CY4386canavanine

[0474] In each case an antagonist should cause the test strain to behavemore like the control strain.

[0475] B. GTPase gα_(s) Mutants (gα Specificity)

[0476] test component=gα_(s)(Q₂₂₇L0

[0477] control component=gα_(s) test control Readout strain effect ofGα_(i2) antagonist strain fus1-HIS3 CY4880 none CY4883 fus1-lacZ CY4886none CY4889 fus2-CAN1 CY4895 none CY4898

[0478] In each case a non-specific antagonist would cause the teststrain to behave more like the control strain.

[0479] Additional media requirements: -TRP for Gα plasmid maintenance infus1-HIS3 and fus2-CAN1 screens and -TRP-URA for Gα and fus1-lacZplasmid maintenance in fus1-lacZ screen.

[0480] II. Suppression of Activation by Receptors

[0481] Constitutively Activated C5a Receptors

[0482] test component=C5aR* (P₁₈₄L, activated C5a Receptor)

[0483] control component=C5aR

[0484] The C5aR* mutation has a Leucine residue in place of the Prolineresidue of the wild-type at position 184 of the amino sequence. testcontrol Readout strain effect of Gα_(i2) antagonist strain fus1-HIS3CY4029 inhibit growth of -HIS + CY2246 AT (Aminotriazole) fus1-lacZCY4901 reduce β-gal activity CY4904 fus2-CAN1 CY4365 induce growth onCY4362 canavanine

[0485] In each case an antagonist should cause the test strain to behavemore like the control strain. Additional media requirements: -LEU forreceptor plasmid maintenance in fus1-HIS3 and fus2-CAN1 screens and-LEU-URA for receptor and fus1-lacZ plasmid maintenance in fus1-lacZscreen, non-buffered yeast media (pH 5.5).

EXAMPLE 9 Identification of a Surrogate Ligand Using Expression of aRandom Peptide Library in Yeast Expressing an Orphan Mammalian Receptor

[0486] FPRL-1 (formyl peptide receptor-like 1) is a structural homologof the formyl peptide receptor (FPR). FPR is a G protein-coupledreceptor, expressed on neutrophils and phagocytic cells, that isstimulated by N-formyl peptides of bacterial origin. Specific binding ofthe natural ligand, f-Met-Leu-Phe, stimulates transduction of a signalto mobilize calcium, resulting in cellular changes including chemotaxisand the release of granule contents. Low stringency hybridization ofHL60 cDNA libraries with an FPR cDNA probe permitted the identificationof the related receptor, FPRL-1 (Murphy et al. supra; Ye et al. supra).The FPRL-1 cDNA encodes a 351 amino acid protein with 69% sequencehomology to FPR (Murphy et al. supra) FPR and FPRL-1 were found toco-localize to human chromosome 19 and to have a tissue expressionpattern identical to that of FPR, i.e., expression is restricted tocells of myeloid origin (Murphy et al. supra). Ye et al. (supra)demonstrated weak binding of f-Met-Leu-Phe (uM concentrations) tofibroblasts transfected with FPRL-1 cDNA. In contrast, Murphy et al.(supra) could not detect binding of N-formyl peptides to Xenopus oocytestransfected with FPRL-1 cDNA. FPRL-1 appears to be an orphan receptorwhose specific ligand differs from the formyl peptide ligands to whichFPR responds.

[0487] In this example experiments detailing the following will bedescribed: (1) establishment of a strain of yeast designed to expressthe human orphan G protein-coupled receptor FPRL-1; (2) expression of arandom peptide library in the aforementioned strain of yeast; and (3)activation of the endogenous yeast pheromone pathway upon stimulation ofthe FPRL-1 receptor by a peptide encoded by a random library expressedwithin the same strain of yeast.

[0488] Preparation of FPRL-1 Yeast Expression Vector

[0489] A plasmid, pFPRL1-L31, containing a 2.6 kb EcoRI-XhoI fragmentencoding the FPRL-1 cDNA in the BluescriptIISK+ vector was obtained fromPhilip Murphy (NIH). The sequence encoding FPRL1 was amplified by thepolymerase chain reaction using VENT polymerase (New England Biolabs,Inc., Beverly, Mass.) through 20 cycles and the followingoligonucleotide primers: #1 5′GGCGCCCGGTCTCCCATGGAAACCAACTTCTCCACT #25′GGCGCCCGGTCTCCGATCCCATTGCCTGTAACTCAGTCTC

[0490] The PCR product was purified, restricted with BsaI and clonedinto Cadus 1651 (p1PBX-1), a PGK promoter-driven expression vector,using NcoI and BamHI sites, to yield CADUS 2311. The sequence of theentire insert was determined and found to be identical to the FPRL-1sequence deposited in GenBank (accession number M84562).

[0491] Preparation of Random Oligonucleotides

[0492] Library-Recycling Protocol to Identify a Surrogate Ligand

[0493] The yeast strain CY1141 (MATalpha far1*1441 tbt1-1 fus1-HIS3 can1 ste14::trp1:;LYS2 ste3*1156 gpa1(41)-Galphai2 lys2 ura3 leu2 trp1his3) was used in the experiments that follow. CY1141 contains apheromone inducible HIS3 gene, fus1-HIS3 integrated at the FUS1 locusand a hybrid gene encoding the first 41 amino acids of GPA1 (yeast Galpha) fused to sequence encoding human G alphai2 (lacking codonsencoding the N-terminal 33 amino acids) replacing GPA1 at itschromosomal locus. The yeast STE14 gene is disrupted to lower the basallevel of signaling through the pheromone response pathway. The yeasta-factor receptor gene, STE3, is deleted. CY 1141 was transformed withCadus 2311 to yield CY6571, a strain expressing the human orphanreceptor, FPRL-1.

[0494] CY6571 exhibited LIRMA (ligand independent receptor mediatedactivation), that is, activation of the yeast pheromone pathway in theabsence of ligand. It was determined that the yeast growth on selectivemedia that resulted from LIRMA was eliminated by the additional of2.5millimolar concentrations of 3-aminotriazole (AT). AT is an inhibitorof the HIS3 gene product that serves to reduce background growth.Therefore, selection protocols aimed at the identification of surrogateligands for the FPRL-1 receptor were carried out at this concentrationof AT.

[0495] CY6571 was inoculated to 10 mls of standard synthetic media (SD)lacking leucine (-Leu) and incubated overnight at 30° C. The 10 mlovernight culture was used to inoculate 50 mls of YEPD; this culture wasincubated at 30° C. for 4.5-5 hours at which time the cells wereharvested and prepared for transformation with DNA encoding a randompeptide library [alpha-NNK (6.24.94)] encoding tridecapeptides of randomsequence, by electroporation. Post electroporation (in 0.2 cm cuvettes,0.25 μF, 200Ω, 1.5 kV) the cells were immediately diluted in 1 mlice-cold 1 M sorbitol and 100 μL aliquots were placed onto 10 syntheticmedia plates (pH 6.8) lacking leucine and uracil (-Leu-Ura). The plateswere incubated at 30° C. for 2-4 days at which time two replicas of eachoriginal transformation plate were made to synthetic media (pH 6.8)lacking leucine, uracil and histidine and supplemented with 2.5 mMAT(-Leu-Ura-His+2.5 mM AT). The replicas were incubated at 30° C. for3-5 days. Post incubation the colonies present on the replica sets oftwo were scraped from the plates into a total of 10 mls of H₂O (5 mlseach plate). The OD₆₀₀ of each cell suspension was determined and crudeplasmid isolations were done on 8-16 OD units of cells for each pool. Atotal of eight pools resulted, due to lower numbers of yeast coloniespresent in four sets of plates. The pellets obtained from these crudeplasmid isolations (the so called “smash and grab” technique, Methods inYeast Genetics—A Laboratory Manual, 1990, M. D. Rose, F. Winston and P.Heiler. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),were resuspended in 40 μL of 10 mM Tris, 1 mM EDTA, pH 8.0 and 1 μL wasused to transform E. coli by electroporation (0.1 cm cuvettes, 0.25 μF,200Ω, 1.8 kV). Post electroporation the cells were immediately dilutedinto 1 ml 2×YT media and incubated, with shaking, at 37° C. for 30minutes after which time the cells were used to inoculate 50 mls of 2×YTsupplemented with 100 ug/ml ampicillin. The 10 resulting cultures wereincubated at 37° C. overnight. Plasmid DNA was isolated from each ofthese bacteria cultures using Qiagen columns (Qiagen, Inc., Chatsworth,Calif.)). Each plasmid DNA pellet was resuspended in 50 μL Tris 10 mM,EDTA 1 mM, pH 8.0.

[0496] Strain CY6571 was transformed with 1 μL of each plasmid pool byelectroporation. Post electroporation the cells were diluted into 400L 1M sorbitol. From each electroporated cell suspension, 1 μL and 400 μL ofcells were plated on -Leu-Ura synthetic media, pH 6.8 to yield “lowdensity” and “high density” platings. The plates were incubated at 30°C. for 3 days, at which time replicas of both the low and high densityplates were made to -Leu-Ura-His+2.5 mM AT. For those cases whereenrichment for a plasmid capable of conferring a His+ phenotype hadoccurred, this would be reflected by an amplified number of His+colonies on both the low and high density plates visible at days 2-3,although the amplification would be most obvious on the plates that hadreceived a high density of cells. In the FPRL-1 experiment 1/8 poolsshowed amplification of His+ colonies. The cells were scraped from thisplate into 5 mls of H₂O, the OD₆₀₀ of the cell suspension was determinedand a crude plasmid isolation was done on 15 OD units of yeast cells.The pellet obtained was resuspended in 40 μL 10 mM Tris, 1 mM EDTA, pH8.0 and 1 μL was used to transform E. coli. Plasmid DNA was isolated byminiprep from 3 ml 2XYT cultures of single bacterial colonies resultingfrom this transformation. 10 DNA pellets (A1 through A10) deriving fromindividual bacterial colonies were resuspended in 20 μL 10 mM Tris 1 mMEDTA, pH 8.0 and used to transform CY6571 (containing the FPRL-1expression vector) and CY6263 (CY1141 containing a control expressionvector lacking any receptor sequence) by electroporation. Cadus 1625, acontrol vector lacking sequences encoding a peptide, was included andused to transform both the receptor+and receptor-strains of yeast.Transformants were first selected on -Leu-Ura, pH 6.8 then three yeasttransformants of each type (from 11 CY6571 transformations and 11 CY6263transformations) were patched to -Leu-Ura, pH 6.8 to expand thecolonies. Once expanded, streaks of the transformants were made on-Leu-Ura-His+2.5 mM AT to test for growth in the absence of histidine.All plasmids except the one denoted A2 conferred a growth advantage onmedia lacking histidine to yeast bearing the FPRL-1-encoding plasmid butnot to yeast lacking the receptor plasmid. The peptide sequence found tobe encoded by plasmids A1 and A3-A10 is:SerLeuLeuTrpLeuThrCysArgProTrpGluAlaMet, and is encoded by thenucleotide sequence 5′-TCT CTG CTT TGG CTG ACT TGT CGG CCT TGG GAG GCGATG-3′.

[0497] Activation of the Pheromone Response Pathway in Yeast Expressingthe FPRL-1 Receptor and Peptide Agonist.

[0498] For verificatiin of pheromone pathway activation andquantification of the stimulation, the activity of the fus1 promoter wasdetermined colorimetrically using a fus1-lacZ fusion in a parallel setof test strains. CY1141, described above, was used as the recipientstrain for these experiments. Transformants contained CADUS 1584(pRS424-fus1-lacZ) in addition to receptor (R^(+/−)) and ligand(L^(+/−)) plasmids. Four strains (bearing the identical plasmids) weregrown overnight in minimal media lacking leucine, uracil, andtryptophan, pH 8.6. The overnight cultures were used to inoculate-Leu-Ura-Trp pH 6.8 media and these new cultures were grown forapproximately 4.5-5 hours to an OD₆₀₀ of less than 0.4. Assay ofβ-galactosidase activity (Guarente 1983) in cells from these culturesyielded the following results: CY1141/CADUS 2311/peptide A1/CADUS 1584R⁺L⁺  28 units CY1141/CADUS 2311/CADUS 1625/CADUS 1584 R⁺L⁻   3 unitsCY1141/CADUS 1289/peptide A1/CADUS 1584 R⁻L⁺ 3.5 units CY1141/CADUS1289/CADUS 1625/CADUS 1584 R⁻L⁻ 3.9 units

[0499] The presence of receptor and peptide-encoding plasmids resultedin an average 8-fold stimulation over background levels ofβ-galactosidase.

[0500] Autocrine Activation of the Pheromone Response Pathway in YeastExpressing by FPRL-1 Agonists or C5a Receptor Agonists.

[0501] The results illustrated in FIG. 11 were obtained using yeastcells engineered to express FPRL-1 or the C5a receptor under conditionswherein the signal transduction from the heterologous receptor wascoupled to a fus1:lacZ reporter gene construct described above. FIG. 11demonstrates the specificity of the surrogate ligand A5 for FPRL-1, andthe surrogate ligand F6, as well as that of the native C5a ligand, forthe C5a receptor. In each instance, the presence of both the receptorand surrogate peptide result in an 8-12 fold increase in lacZ expressionover the level observed in the absence of either the receptor, ligand,or both.

[0502] Activation of Human Neutrophils by a Surrogate FPRL Agonist.

[0503] Human neutrophils in culture were stimulated with varyingconcentrations of the FPRL surrogate ligand A5, and intracellular Ca⁺⁺mobilization was detected by Fluorescence Activated Cell Sorter (FACS)analysis based on FURA2 dye absorbance ratios. The response of the humanneutrophils to the C5a peptide was also measured. As shown in FIG. 12,the A5 peptide produced a dose-dependent increase in intracellularcalcium mobilization, indicating that it is capable of activatingendogenous FPRL-mediated pathways in human neutrophils.

[0504] Preparation of Second Generation FPRL Ligand Libraries.

[0505] To further improve the selectivity and/or potency of the agonistsidentified by the above steps, we selected a surrogate peptide (A5), andcreated degenerate peptide libraries based on the sequence of thatpeptide as a starting point (e.g., a semi-random library) as follows:FPRL-1 peptide A5 Ser-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Metsub-libraries N-term Xaa-Xaa-Xaa-Xaa-Xaa-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Metmid4 Ser-Leu-Leu-Trp-Leu-Xaa-Xaa-Xaa-Xaa-Trp-Glu-Ala-Met C-termSer-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Xaa-Xaa-Xaa-Xaa mod2Xaa-Leu-Xaa-Trp-Xaa-Thr-Xaa-Arg-Xaa-Trp-Xaa-Ala-Xaa mod3Ser-Xaa-Leu-Xaa-Leu-Xaa-Cys-Xaa-Pro-Xaa-Glu-Xaa-Met

[0506] Following the protocols set out above for the first generationpeptide library, the second generation peptide library was screened, andindividual clones isolated based on their ability to stimulate FPRLreceptor dependent transcription.

EXAMPLE 10 Identification of Surrogate Ligands Using Expression of aRandom Peptide Library in Yeast Expressing the Orphan MammalianReceptor, MDR-15.

[0507] In a similar manner a plasmid encoding the monocyte derivedreceptor monocyte-derived receptor 15 (MDR15; Barella et al. (1995)Biochem. J 309:773-9) was used to construct a yeast strain (CY6573)expressing this receptor. This receptor is an alternative spliced formof the Burkitt's lymphoma receptor 1 (BLR1) encoded by a human Burkitt'slymphoma cDNA (Dobner et al. (1992) Eur. J Immunol. 22, 2795-2799).Strain CY6573 was transformed in a similar manner with the NNK13library, and, following selection on ten -Leu-Ura (4.4×10⁵ colonies perplate), replica plated to -Leu-Ura-His+1 mM AT plates. Upon reisolationof plasmid pools and re-transformation into strain CY6573; eight of tenpools showed signicantly enriched colony formation on -Leu-Ura-His+1 mMAT plates. Eight unique plasmids derived from these pools whenretransformed into CY6573 conferred growth on -Leu-Ura-His+1 mM ATplates. One of these plasmids failed to confer growth in a yeast strainlacking the MDR15 receptor.

EXAMPLE 11 Identification of a Ligand Using Expression of a RandomPeptide Library in Yeast Expressing the Human Thrombin Receptor

[0508] The receptor for thrombin, a G protein-coupled receptor, ispresent on numerous cell types including platelets, vascular smoothmuscle, fibroblasts and on a subset of cells that function in immunity.Thrombin, a serine protease, binds to and cleaves the receptor moleculeat residue 41, generating a new receptor N-terminus. The post-cleavageN-terminal residues then act as a “tethered ligand’ to activate thereceptor molecule (Vu et al. 1994). In platelets, signaling through thethrombin receptor has been shown to result in numerous effects includingstimulation of phospholipase C, mobilization of intracellular Ca²⁺ andinhibition of adenylyl cyclase.

[0509] In this example experiments that detail the following will bedescribed (1) establishment of a strain of yeast designed to express thehuman G protein-coupled receptor for thrombin; (2) expression of arandom peptide library in the afore-mentioned strain of yeast and (3)activation of the endogenous yeast pheromone pathway upon stimulation ofthe thrombin receptor by peptides encoded by a random library expressedwithin the same strain of yeast.

[0510] Preparation of a Yeast Expression Vector for a Mammalian ThrombinReceptor

[0511] The human thrombin receptor was amplified by PCR frompcDNA3:Hu-Thr9b-5′ (Bristol Myers Squibb) using the followingoligonucleotides: 5′ GGGCCATGGGGCCGCGGCGGTTG 3′5′ CCCGGATCCTAAGTTAACAGCTTTTTGTATAT 3′

[0512] The amplified product was purified by gel electrophoresis,restricted with NcoI and BamHII and ligated to NcoI and BamHI-cut CADUS1871, a PGK promoter-driven expression vector, to yield CADUS 2260.Cloning into CADUS 1871 introduces a novel stop codon preceded by thetriplet GlySerVal after the authentic carboxy terminal codon of thehuman thrombin receptor (threonine). In addition, an invertase signalsequence is fused to the authentic amino terminus of the receptor.

[0513] CY7467 exhibited LIRMA (ligand independent receptor mediatedactivation), that is, activation of the yeast pheromone pathway in theabsence of ligand. It was determined that the yeast growth on selectivemedia that resulted from LIRMA was eliminated by the addition of 2.5millimolar concentrations of 3-aminotriazole (AT). AT is an inhibitor ofthe HIS3 gene product that serves to reduce background growth.Therefore, selection protocols aimed at the identification of novelpeptide ligands for the human thrombin receptor were carried out at thisconcentration of AT.

[0514] Preparation of Random Oligonucleotide Library

[0515] As described above.

[0516] Recycling Protocol to Identify a Surrogate Ligand

[0517] The yeast strain CY1141 (MATalpha far1*1442 tbt1-1 fus1-HIS3 can1ste14::trp1::LYS2 ste3*1156 gpa1(41)-Galphai2 lys2 ura3 leu2 trp1 his3)was transformed with CADUS 2260 to yield strain CY7467, expressing thehuman thrombin receptor. CY7467 was inoculated to 10 mls of standardsynthetic media (SD) lacking leucine (-Leu) and incubated overnight at30 C. The 10 ml overnight culture was used to inoculate 50 mls of YEPDmedia; this culture was incubated at 30 C for 4.5-5 hours at which timethe cells were harvested and prepared for transformation with DNAencoding a random peptide library [alpha-NNK (6.24.94)] byelectroporation. Post electroporation (in 0.2 cm cuvettes, 0.25 mF,200W, 1.5 kV) the cells were immediately diluted in 1 ml ice-cold 1Msorbitol and 100 mL aliquots were plated onto 10 synthetic media plates(pH 6.8) lacking leucine and uracil (-Leu-Ura). The plates wereincubated at 30 C for 2-4 days at which time two replicas of eachoriginal transformation plate were made to synthetic media (pH 6.8)lacking leucine, uracil and histidine and supplemented with 2.5 mMAT(-Leu-Ura-His+2.5 mM AT). The replicas were incubated at 30 C for 3-5days. Post incubation the colonies present on the replica sets of twowere scraped from the plates into a total of 10 mls of H₂O (5 mls eachplate). The OD₆₀₀ of each cell suspension was determined and crudeplasmid isolations were done on 8-16 OD units of cells for each pool. Atotal of ten pools resulted. The pellets obtained from these crudeplasmid isolations were resuspended in 40 mL of 10 mM Tris, 1 mM EDTA,pH 8.0 and 1 mL was used to transform E. coli by electroporation (0.1 cmcuvettes, 0.25 mF, 200W, 1.8 kV). Post electroporation the cells wereimmediately diluted into 1 ml 2XYT media and incubated, with shaking, at37 C for 30 minutes after which time the cells were used to inoculate 50mls of 2xYT supplemented with 100 ug/ml ampicillin. The 10 resultingcultures were incubated at 37 C overnight. Plasmid DNA was isolated fromeach of these bacterial cultures using Qiagen columns (Qiagen, Inc.,Chatsworth, Calif.). Each plasmid DNA pellet was resuspended in 50 mLTris 10 mM, EDTA 1 mM, pH 8.0.

[0518] Strain CY7467 was transformed with 1 mL of each plasmid pool byelectroporation. Post electroporation the cells were diluted into 400 mLIM sorbitol. From each electroporated cell suspension, 1 mL and 400 mLof cells were plated on -Leu-Ura synthetic media, pH 6.8 to yield “lowdensity” and “high density” platings. The plates were incubated at 30 Cfor 3 days, at which time replicas of both the low and high densityplates were made to -Leu-Ura-His+2.5 mM AT. For those cases whereenrichment for a plasmid capable of conferring a His+ phenotype hadoccurred, this would be reflected by an amplified number of His+colonies on both the low and high density plates visible at days 2-3,although the amplification would be most obvious on the plates that hadreceived a high density of cells. In this experiment 3/10 pools showedamplification of His+ colonies. The cells from each of these plates werescraped into 5 mls of H₂O, the OD₆₀₀ of the cell suspensions weredetermined and crude plasmid isolations were done on 8-16 OD units ofyeast cells. The pellets obtained were resuspended in 40 mL 10 mM Tris,1 mM EDTA, pH 8.0 and 1 mL was used to transform E. coli. Plasmid DNAwas isolated by miniprep from 3 ml 2XYT cultures of single bacterialcolonies resulting from these transformations (three bacterial coloniesfor each DNA pool were processed in this way). DNAs deriving from threeindividual bacterial colonies per pool were resuspended in 20 mL 10 mMTris 1 mM EDTA, pH 8.0. The three DNAs derived per pool were sequencedand found to encode identical peptides. Thus three differing DNAsequences were derived, one representing each amplified pool. Oneplasmid representing each of the three original amplified pools was usedto transform CY7467 (containing the thrombin receptor expression vector)and CY6263 (CY 1141 containing a control expression vector lacking anyreceptor sequence) by electroporation. CADUS 1625, a control vectorlacking sequences encoding a peptide was included and used to transformboth the receptor+ and receptor− strains of yeast. CADUS 1651, a controlvector lacking sequences encoding a receptor included and used totransform both the ligand+ and ligand− strains of yeast. Transformantswere first selected on -Leu-Ura, pH 6.8, then two yeast transformants ofeach type were patched to -Leu-Ura, pH 6.8 to expand the colonies. Onceexpanded, streaks of the transformants were made on -Leu-Ura-His+2.5 mMAT to test for growth in the absence of histidine. One of the threeplasmids tested conferred a growth advantage on media lacking histidineto yeast bearing the thrombin-encoding plasmid but not to yeast lackingthe receptor plasmid. The peptide sequence encoded by this plasmid is:Val-Cys-Pro-Ala-Arg-Tyr-Val-Leu-Pro-Gly-Pro-Val-Leu and was encoded bythe nucleotide sequence GTT TGT CCT GCG CGT TAT GTG CTG CCT GGG CCT GTTTTG. TABLE 1 Detection of C5a production in yeast by ELISA. R−L− R+L−R−L+ R+L+ [C5a] in n.d. n.d. 0.64 ng/ml = 77 nM 0.5 ng/ml = 60 nMculture [C5a] released n.d. n.d.  0.8 ng/ml = 97 nM 0.6 ng/ml = 73 nMfrom lysed cells*

[0519] TABLE 2 Coupling of the C5a receptor to Gα chimeras in yeast.Expression Chimera Context Result GPA1₄₁-Gαi2 single copy, Good signalto noise ratio: integrated, efficient coupling to yeast GPA1 promoterβγ. GPA1₄₁-Gαi3 single copy, Poor signal to noise ratio: integrated,high background due to poor GPA1 promoter coupling to yeast βγ, highLIRMA*. GPA1βam-Gαi2 low copy plasmid, Signal equal to that with GPA 1promoter GPA1₄₁-Gαi2, however, back- ground is greater. GPA1βam-Gαl6 lowcopy plasmid, Poor signal to noise ratio, GPA1 promoter high backgrounddue to poor coupling to yeast βγ, high LIRMA*. GPA1βam-Gαs low copyplasmid, Unacceptably high background GPA1 promoter due to poor couplingto yeast βγ, high LIRMA*. # transgenic mice.

[0520] LIRMA may be exploited in several ways, including theidentification of antagonists capable of reducing the phenomenon. Asubset of antagonists would be expected to affect the receptorconformation in such a way as to prevent the downstream signalling thatoccurs in the absence of agonist. LIRMA can be exploited to identify newG protein-coupled receptors by expressing cDNA clones in yeast strainsexpressing those chimeric G proteins which couple only poorly to yeastβγ. In addition, LIRMA may permit the identification of inhibitors thatare specific for G proteins. TABLE 3 Coupling of Gα switch regionhybrids to the pheromone response pathway. GPA1 amino Gas amino acidProtein acid sequences sequences Phenotype GPA1  1-472 none Couples withGβγ GαS none  1-394 Couples with Gβγ weakly GPA₄₁-S  1-41 42-394 Coupleswith Gβγ weakly SGS 297-333  1-201 + 237-394 Does not couple with GβγGPA₄₁-SGS 1-41 + 297-333 42-201 + 237-394 Couples with Gβγ weakly

We claim:
 1. A mixture of recombinant cells, each cell of whichcomprises: (i) an expressible recombinant gene encoding a heterologousreceptor protein whose signal transduction activity is modulated byinteraction with an extracellular signal; and (ii) an expressiblerecombinant gene encoding a heterologous potential receptor effectorpolypeptide, wherein collectively the mixture of cells expresses avariegated population of said receptor effector polypeptides, andmodulation of the signal transduction activity of the receptor proteinby a test polypeptide provides a detectable signal.
 2. A mixture ofrecombinant cells, each cell of which comprises: (i) a heterologousreceptor protein whose signal transduction activity is modulated byinteraction with an extracellular signals; (ii) an expressiblerecombinant gene encoding a heterologous potential receptor effectorpolypeptide; and (iii) a reporter gene construct containing a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the signal transduction acitivity of the receptorprotein, wherein collectively the mixture of cells expresses avariegated population of test polypeptides as receptor effectors.
 3. Thecells of claim 2, wherein the receptor is a nuclear receptor.
 4. Thecells of claim 2, wherein the receptor is a cell surface receptor.
 5. Amixture of recombinant cells, each cell of which comprises: (i) areceptor protein whose signal transduction activity is modulated byinteraction with an extracellular signals; (ii) an expressiblerecombinant gene encoding a heterologous potential receptor effectorpolypeptide; and (iii) a reporter gene construct containing a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the signal transduction acitivity of the receptorprotein, wherein collectively the mixture of cells expresses avariegated population of test polypeptides as receptor effectors.
 6. Thecells of claim 5, wherein the receptor is a nuclear receptor.
 7. Thecells of claim 5, wherein the receptor is a cell surface receptor.
 8. Amixture of recombinant cells, each cell of which comprises: (i) a cellsurface receptor protein whose signal transduction activity is modulatedby interaction with an extracellular signal; and (ii) an expressiblerecombinant gene encoding a heterologous potential receptor effectorpolypeptide including a signal sequence for secretion, whereincollectively the mixture of cells expresses a variegated population oftest polypeptides as receptor effectors, and modulation of the signaltransduction activity of the receptor protein by a test polypeptideprovides a detectable signal.
 9. The recombinant cells of claim 8,wherein each cell further comprises a reporter gene construct containinga reporter gene in operative linkage with one or more transcriptionalregulatory elements responsive to the signal transduction acitivity ofthe cell surface receptor protein, expression of the reporter geneproviding the detectable signal.
 10. The recombinant cells of claim 8,wherein the reporter gene encodes a gene product that gives rise to adetectable signal selected from the group consisting of: color,fluorescence, luminescence, cell viability relief of a cell nutritionalrequirement, cell growth, and drug resistance.
 11. The recombinant cellsof claim 9, wherein the reporter gene encodes a gene product selectedfrom the group consisting of chloramphenicol acetyl transferase,beta-galactosidase and secreted alkaline phosphatase.
 12. Therecombinant cells of claim 9, wherein the reporter gene encodes a geneproduct which confers a growth signal.
 13. The recombinant cells ofclaim 9, wherein the reporter gene encodes a gene product for growth inmedia containing aminotriazole or canavanine.
 14. The recombinant cellsof claim 8, wherein the detectable signal comprises intracellularcalcium mobilization.
 15. The recombinant cells of claim 8, wherein thedetectable signal comprises a 1 significant change in intracellularprotein phosphorylation.
 16. The recombinant cells of claim 8, whereinthe detectable signal comprises increases in phospholipid metabolism.17. The recombinant cells of claim 8, wherein each cell furthercomprises a heterologous gene construct encoding the receptor protein.18. The recombinant cells of claim 8, wherein the receptor protein is aG-protein coupled receptor.
 19. The recombinant cells of claim 18,wherein the G-protein coupled receptor is selected from the groupconsisting of: a chemoattractant peptide receptor, a neuropeptidereceptor, a light receptor, a neurotransmitter receptor, a cyclic AMPreceptor, and a polypeptide hormone receptor.
 20. The recombinant cellsof claim 8 wherein the receptor protein is a receptor tyrosine kinase.21. The recombinant cells of claim 20, wherein the receptor tyrosinekinase is an EPH receptor.
 22. The recombinant cells of claim 8, whereinthe receptor protein is an orphan receptor.
 23. The recombinant cells ofclaim 8, which recombinant cells are yeast cells.
 24. The recombinantcells of claim 8, which recombinant cells are mammalian cells.
 25. Therecombinant cells of claim 8, wherein the variegated population of testpolypeptides includes at least 10³ different test polypeptides.
 26. Arecombinant cell, comprising: (i) an expressible recombinant geneencoding a heterologous cell surface receptor protein whose signaltransduction activity is modulated by extracellular signals; (ii) anexpressible recombinant gene encoding a heterologous potential receptoreffector polypeptide including a signal sequence for secretion; and(iii) a reporter gene construct containing a reporter gene in operativelinkage with one or more transcriptional regulatory elements responsiveto the signal transduction acitivity of the cell surface receptorprotein.
 27. The recombinant cell of claim 26, wherein the reporter geneencodes a gene product that gives rise to a detectable signal selectedfrom the group consisting of: color, fluorescence, luminescence, cellviability relief of a cell nutritional requirement, cell growth, anddrug resistance.
 28. The recombinant cell of claim 26, wherein thereceptor protein is a G-protein coupled receptor.
 29. The recombinantcell of claim 28, wherein the G-protein coupled receptor is selectedfrom the group consisting of: a chemoattractant peptide receptor, aneuropeptide receptor, a light receptor, a neurotransmitter receptor, acyclic AMP receptor, and a polypeptide hormone receptor.
 30. Therecombinant cell of claim 26, wherein the receptor protein is a receptortyrosine kinase.
 31. The recombinant cell of claim 30, wherein thereceptor tyrosine kinase is an EPH receptor.
 32. The recombinant cell ofclaim 26, wherein the receptor protein is an orphan receptor.
 33. Therecombinant cell of claim 26, wherein the receptor protein is a cytokinereceptor.
 34. The recombinant cell of claim 26, wherein the receptorprotein is an MIRR.
 35. The recombinant cell of claim 26, whichrecombinant cell is a yeast cell.
 36. The recombinant cell of claim 35,which yeast cells is a Saccharomyces cell.
 37. The recombinant cell ofclaim 35, which yeast cells is a Schizosaccharomyces cell.
 38. Therecombinant cell of claim 26, which cells are mammalian cells.
 39. Amixture of recombinant cells, each cell of which comprises: (i) anexpressible recombinant gene encoding a heterologous cell surfacereceptor protein whose signal transduction activity is modulated byextracellular signals; (ii) an expressible recombinant gene encoding aheterologous potential receptor effector polypeptide including a signalsequence for secretion; and (iii) a reporter gene construct containing areporter gene in operative linkage with one or more transcriptionalregulatory elements responsive to the signal transduction acitivity ofthe cell surface receptor protein, wherein collectively the mixture ofcells expresses a variegated population of test polypeptides.
 40. Therecombinant cells of claim 39, wherein the receptor protein is aG-protein coupled receptor.
 41. The recombinant cells of claim 40,wherein the G-protein coupled receptor is selected from the groupconsisting of: a chemoattractant peptide receptor, a neuropeptidereceptor, a light receptor, a neurotransmitter receptor, a cyclic AMPreceptor, and a polypeptide hormone receptor.
 42. The recombinant cellof claim 40, wherein the G-protein coupled receptor is selected from thegroup consisting of: α1A-adrenergic receptor, α1B-adrenergic receptor,α2-adrenergic receptor, α2B-adrenergic receptor, β1-adrenergic receptor,β2-adrenergic receptor, β3-adrenergic receptor, ml acetylcholinereceptor (AChR), m2 AChR, m3 ACHR, m4 AChR, m5 AChR, D1 dopaminereceptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopaminereceptor, D5 dopamine receptor, A1 adenosine receptor, A2b adenosinereceptor, 5-HT1a, 5-HT1b, 5HT1-like, 5-HT1d, 5HT1d-like, 5HT1d beta,substance K (neurokinin A), fMLP receptor, fMLP-like receptor,angiotensin II type 1, endothelin ETA, endothelin ETB, thrombin, growthhormone-releasing hormone (GHRH), vasoactive intestinal peptide,oxytocin, somatostatin SSTR1 and SSTR2, SSTR3, cannabinoid, folliclestimulating hormone (FSH), leutropin (LH/HCG), thyroid stimulatinghormone (TSH), thromboxane A2, platelet-activating factor (PAF), C5aanaphylatoxin, Interleukin 8 (IL-8) IL-8RA, IL-8RB, Delta Opioid, KappaOpioid, mip-1/RANTES, Rhodopsin, Red opsin, Green opsin, Blue opsin,metabotropic glutamate mGluR1-6, histamine H2, ATP, neuropeptide Y,amyloid protein precursor, insulin-like growth factor II, bradykinin,gonadotropin-releasing hormone, cholecystokinin, melanocyte stimulatinghormone receptor, antidiuretic hormone receptor, glucagon receptor, andadrenocorticotropic hormone II.
 43. The recombinant cells of claim 39,wherein the receptor protein is a receptor tyrosine kinase.
 44. Therecombinant cells of claim 43, wherein the receptor tyrosine kinase isan EPH receptor.
 45. The yeast cell of claim 44, wherein the receptor isselected from the group consisting of: eph, elk, eck, sek, mek4, hek,hek2, eek, erk, tyro1, tyro4, tyro5, tyro6, tyro11, cek4, cek5, cek6,cek7, cek8, cek9, cek10, bsk, rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2,pagliaccio, htk, erk and nuk receptors.
 46. The recombinant cell ofclaim 39, wherein the receptor protein is a cytokine receptor.
 47. Therecombinant cell of claim 39, wherein the receptor protein is an MIRRreceptor.
 48. The recombinant cell of claim 39, wherein the receptorprotein is an orphan receptor.
 49. The recombinant cell of claim 39,which recombinant cell is a yeast cell.
 50. The recombinant cell ofclaim 49, which yeast cells is a Saccharomyces cell.
 51. The recombinantcell of claim 49, which yeast cells is a Schizosaccharomyces cell. 52.The recombinant cell of claim 39, which cells are mammalian cells. 53.The recombinant cells of claim 39, wherein the variegated population oftest polypeptides includes at least 10³ different test polypeptides. 54.A method for identifying potential receptor effectors comprising: (i)providing a mixture of recombinant cells, each cell of which comprises(a) a receptor protein whose signal transduction activity is modulatedby interaction with an extracellular signal, and (b) an expressiblerecombinant gene encoding a heterologous test polypeptide. wherein themixture of cells collectively express a variegated population of testpolypeptides, and modulation of the signal transduction activity of thereceptor protein by a test polypeptide provides a detection signal; and(ii) isolating cells from the mixture which exhibit the detectionsignal.
 55. The method of claim 54, wherein the cell receptor is a cellsurface receptor.
 56. The method of claim 55, wherein the heterologoustest polypeptide includes a signal sequence for secretion.
 57. Themethod of claim 54, wherein each cell of the mixture further comprises areporter gene construct containing a reporter gene in operative linkagewith one or more transcriptional regulatory elements responsive to thesignal transduction acitivity of the cell surface receptor protein,expression of the reporter gene providing the detection signal.
 58. Themethod of claim 57, wherein the reporter gene encodes a gene productthat gives rise to a detection signal selected from the group consistingof: color, fluorescence, luminescence, cell viability relief of a cellnutritional requirement, cell growth, and drug resistance.
 59. Themethod of claim 58, wherein the reporter gene encodes a gene productselected from the group consisting of chloramphenicol acetyltransferase, beta-galactosidase and secreted alkaline phosphatase. 60.The method of claim 58, wherein the reporter gene encodes a gene productwhich confers a growth signal.
 61. The method of claim 58, wherein thereporter gene encodes a gene product for growth in media containingaminotriazole or canavanine.
 62. The method of claim 54, wherein thedetection signal comprises intracellular calcium mobilization.
 63. Themethod of claim 54, wherein the detection signal comprises astatistically significant change in intracellular proteinphosphorylation.
 64. The method of claim 54, wherein the detectionsignal comprises changes in phospholipid metabolism.
 65. The method ofclaim 54, wherein each cell of the mixture further comprises aheterologous gene construct encoding the receptor protein.
 66. Themethod of claim 54, wherein the receptor protein is a G-protein coupledreceptor.
 67. The method of claim 66, wherein the G-protein coupledreceptor is selected from the group consisting of: a chemoattractantpeptide receptor, a neuropeptide receptor, a light receptor, aneurotransmitter receptor, a cyclic AMP receptor, and a polypeptidehormone receptor.
 68. The method of claim 54, wherein the receptorprotein is a receptor tyrosine kinase.
 69. The method of claim 68,wherein the receptor tyrosine kinase is an EPH receptor.
 70. The methodof claim 54, wherein the receptor protein is a cytokine receptor. 71.The method of claim 54, wherein the receptor protein is an orphanreceptor.
 72. The method of claim 54, which recombinant cells are yeastcells.
 73. The method of claim 54, which recombinant cells are mammaliancells.
 74. The method of claim 54, wherein the variegated population oftest polypeptides includes at least 10³ different test polypeptides. 75.A method for identifying effectors of a cell surface receptorcomprising: (i) providing a mixture of recombinant cells, each cell ofwhich comprises (a) an expressible recombinant gene encoding aheterologous cell surface receptor protein whose signal transductionactivity is modulated by extracellular signals, (b) an expressiblerecombinant gene encoding a heterologous potential receptor effectorpolypeptide including a signal sequence for secretion, and (c) areporter gene construct containing a reporter gene in operative linkagewith one or more transcriptional regulatory elements responsive to thesignal transduction acitivity of the cell surface receptor protein,wherein the mixture of cells collectively express a variegatedpopulation of test polypeptides, and modulation of the signaltransduction activity of the receptor protein by a test polypeptidecauses a statistically significant change in the level of expression ofthe reporter gene; and (ii) isolating cells from the mixture whichexhibit the detection signal.
 76. A method for identifying ligands foran orphan cell surface receptor comprising: (i) providing a mixture ofrecombinant cells, each cell of which comprises (a) a heterologous geneencoding an orphan cell surface receptor whose signal transductionactivity is modulated by extracellular signals; and (b) an expressiblerecombinant gene encoding a heterologous test polypeptide including asignal sequence for secretion, wherein the mixture of cells collectivelyexpress a variegated population of test polypeptides, and modulation ofthe signal transduction activity of the orphan receptor protein by atest polypeptide provides a detection signal; and (ii) isolating cellsfrom the mixture which exhibit the detection signal.